Methods to prepare conditionally replicating viral vectors

ABSTRACT

The present invention provides a conditionally replicating viral vector, methods of making, modifying, propagating and selectively packaging, and using such a vector, isolated molecules of specified nucleotide and amino acid sequences relevant to such vectors, a pharmaceutical composition and a host cell comprising such a vector, the use of such a host cell to screen drugs. The methods include the prophylactic and therapeutic treatment of viral infection, in particular HIV infection, and, thus, are also directed to viral vaccines and the treatment of cancer, in particular cancer of viral etiology. Other methods include the use of such conditionally replicating viral vectors in gene therapy and other applications.

This application claims priority to U.S. patent application Ser. No.08/563,459, which was filed on Nov. 28, 1995, has since been convertedto provisional patent application Ser. No. 60/032,800, and is herebyincorporated by reference in its entirety.

This application claims priority to U.S. patent application Ser. No.08/563,459, which was filed on Nov. 28, 1995, has since been convertedto provisional patent application Ser. No. 60/032,800, and is herebyincorporated by reference in its entirety.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to a conditionally replicating viralvector, methods of making, modifying, propagating and selectivelypackaging such a vector, isolated molecules of specified nucleotide andamino acid sequences relevant to such vectors, a pharmaceuticalcomposition and a host cell comprising such a vector, and methods ofusing such a vector and a host cell.

BACKGROUND OF THE INVENTION

The discovery of the human immunodeficiency virus (HIV) as the cause ofacquired immune deficiency syndrome (AIDS) has fostered a plethora ofresearch into the underlying mechanisms of the viral infectious cycleand viral pathogenesis. Studies on these mechanisms have providedresearchers with an ever-increasing number of targets for thedevelopment of antiviral agents effective not only against HIV, butagainst other viruses as well. These antiviral agents, particularlythose directed against HIV, can be categorized into groups depending ontheir mode of action. Such groups include inhibitors of reversetranscriptase, competitors of viral entry into cells, vaccines, andprotease inhibitors, as well as a more recent group referred to hereinas "genetic antiviral agents."

Generally, each type of antiviral agent has its own associated benefitsand limitations, and must be assessed in terms of the exigencies of theparticular treatment situation. Antiviral agents, such as zidovudine(3'-azido-3'-deoxythymidine, also known as AZT), protease inhibitors andthe like, can be delivered into the cells of a patient's body withrelative ease and have been studied extensively. Targeting one specificfactor in the viral infectious cycle, such agents have proven relativelyineffective against HIV. This is primarily due to the fact that strainsof HIV change rapidly and become resistant to agents having a singularlocus of effect (Richman, AIDS Res. and Hum. Retrovir., 8, 1065-1071(1992)) Accordingly, the problems of genetic variation and rapidmutation in HIV genomes compel consideration of new antiviral strategiesto treat HIV infections. Along these lines, genetic antiviral agents areattractive, since they work at many different levels intracellularly.

Genetic antiviral agents differ from other therapeutic agents in thatthey are transferred as molecular elements into a target cell, whereinthey protect the cell from viral infection (Baltimore, Nature, 325,395-396 (1988); and Dropulic' et al., Hum. Gene Ther., 5, 927-939(1994)). Genetic antiviral agents can be any genetic sequence andinclude, but are not limited to, antisense molecules, RNA decoys,transdominant mutants, interferons, toxins, immunogens, and ribozymes.In particular, ribozymes are genetic antiviral agents that cleave targetRNAs, including HIV RNA, in a sequence-specific fashion. The specificityof ribozyme-mediated cleavage of target RNA suggests the possible use ofribozymes as therapeutic inhibitors of viral replication, including HIVreplication. Different types of ribozymes, such as the hammerhead andhairpin ribozymes, have been used in anti-HIV strategies (see, e.g.,U.S. Pat. Nos. 5,144,019, 5,180,818 and 5,272,262, and PCT patentapplication nos. WO 94/01549 and WO 93/23569). Both of the hammerheadand hairpin ribozymes can be engineered to cleave any target RNA thatcontains a GUC sequence (Haseloff et al., Nature, 334, 585-591 (1988);Uhlenbeck, Nature, 334, 585 (1987); Hampel et al., Nuc. Acids Res., 18,299-304 (1990); and Symons, Ann. Rev. Biochem., 61, 641-671 (1992)).Generally speaking, hammerhead ribozymes have two types of functionaldomains, a conserved catalytic domain flanked by two hybridizationdomains. The hybridization domains bind to sequences surrounding the GUCsequence and the catalytic domain cleaves the RNA target 3' to the GUCsequence (Uhlenbeck (1987), supra; Haseloff et al. (1988), supra; andSymons (1992), supra).

A number of studies have confirmed that ribozymes can be at leastpartially effective at inhibiting the propagation of HIV in tissueculture cells (see, e.g., Sarver et al., Science, 247, 1222-1225 (1990);Sarver et al., NIH Res., 5, 63-67 (1993a); Dropulic' et al., J. Virol.,66, 1432-1441 (1992); Dropulic' et al., Methods: Comp. Meth. Enzymol.,5, 43-49 (1993); Ojwang et al., PNAS, 89, 10802-10806 (1992); Yu et al.,PNAS, 90, 6340-6344 (1993); and Weerasinghe et al., J. Virol., 65,5531-5534 (1991)). In particular, Sarver et al. ((1990), supra) havedemonstrated that hammerhead ribozymes designed to cleave within thetranscribed region of the HIV gag gene, i.e., anti-gag ribozymes, couldspecifically cleave HIV gag RNAs in vitro. Furthermore, when cell linesexpressing anti-gag ribozymes were challenged with HIV-1, a 50- to100-fold inhibition of HIV replication was observed. Similarly,Weerasinghe et al. ((1991), supra) have shown that retroviral vectorsencoding ribozymes designed to cleave within the U5 sequence of HIV-1RNA confer HIV resistance to transduced cells upon subsequent challengewith HIV. Although different clones of transduced cells demonstrateddifferent levels of resistance to challenge as determined by thepromoter system used to drive ribozyme expression, most of theribozyme-expressing cell lines succumbed to HIV expression after alimited time in culture.

Transduction of tissue culture cells with a provirus into the nef gene(which is not essential for viral replication in tissue culture) ofwhich was introduced a ribozyme, the hybridization domains of which werespecific for the U5 region of HIV, has been shown to inhibit viralreplication within the transduced cells 100-fold as compared to cellstransduced with wild-type proviruses (see, e.g., Dropulic' et al. (1992)and (1993), supra). Similarly, hairpin ribozymes have been shown toinhibit HIV replication in T-cells transduced with vectors containing U5hairpin ribozymes and challenged with HIV (Ojwang et al. (1992), supra).Other studies have shown that vectors containing ribozymes expressedfrom a tRNA promoter also inhibit a variety of HIV strains (Yu et al.(1993), supra).

Delivery of ribozymes or other genetic antiviral agents to the cellulartargets of HIV infection (e.g., CD4+ T-cells and monocytic macrophages)has been a major hurdle for effective genetic therapeutic treatment ofAIDS. Current approaches for targeting cells of the hematopoietic system(i.e., the primary targets for HIV infection) call for introduction oftherapeutic genes into precursor multipotent stem cells, which, upondifferentiation, give rise to mature T-cells, or, alternatively, intothe mature CD4+ T lymphocytes, themselves. The targeting of stem cellsis problematic, however, since the cells are difficult to culture andtransduce in vitro. The targeting of circulating T lymphocytes is alsoproblematic, since these cells are so widely disseminated that it isdifficult to reach all target cells using current vector deliverysystems. Moreover, macrophages need to be considered as a cellulartarget, since they are the major reservoir for viral spread to otherorgans. However, since macrophages are terminally differentiated and,therefore, do not undergo cellular division, they are not readilytransduced with commonly used vectors.

Accordingly, the predominant current approach to HIV treatment makes useof replication-defective viral vectors and packaging (i.e., "helper")cell lines (see, e.g., Buchschacher, JAMA, 269(22), 2880-2886 (1993);Anderson, Science, 256, 808-813 (1992); Miller, Nature, 357, 455-460(1992); Mulligan, Science, 260, 926-931 (1993); Friedmann, Science, 244,1275-1281 (1989); and Cournoyer et al., Ann. Rev. Immunol., 11, 297-329(1993)) to introduce into cells susceptible to viral infection (such asHIV infection) a foreign gene that specifically interferes with viralreplication, or that causes the death of an infected cell (reviewed byBuchschacher (1993), supra). Such replication-defective viral vectorscontain, in addition to the foreign gene of interest, the cis-actingsequences necessary for viral replication but not sequences that encodeessential viral proteins. Consequently, such a vector is unable tocomplete the viral replicative cycle, and a helper cell line, whichcontains and constitutively expresses viral genes within its genome, isemployed to propagate it. Following introduction of areplication-defective viral vector into a helper cell line, proteinsrequired for viral particle formation are provided to the vector intrans, and vector viral particles capable of infecting target cells andexpressing therein the gene, which interferes with viral replication orcauses a virally infected cell to die, are produced.

Such replication-defective retroviral vectors include adenoviruses andadeno-associated viruses, as well as those retroviral vectors employedin clinical trials of HIV gene therapy, and, in particular, the mouseamphotropic retroviral vector known as the Moloney murine leukemia virus(MuLV). These defective viral vectors have been used to transduce CD4+cells with genetic antiviral agents, such as anti-HIV ribozymes, withvarying degrees of success (Sarver et al. (1990), supra; Weerasinghe etal. (1991), supra; Dropulic' et al. (1993), supra; Ojwang et al. (1992),supra; and Yu et al. (1993), supra). However, these vectors areintrinsically limited for HIV gene therapy applications. For example, ahigh transduction frequency is especially important in the treatment ofHIV, where the vector has to transduce either rare CD34+ progenitorhematopoietic stem cells or widely disseminated target CD4+ T-cells,most of which, during the clinical "latent" stage of disease, arealready infected with HIV. MuLV vectors, however, are difficult toobtain in high titer and, therefore, result in poor transduction.Furthermore, long-term expression of transduced DNA has not beenobtained in CD34+ progenitor stem cells, in particular afterdifferentiation to mature T lymphocytes. In addition, the use ofdefective viral vectors requires ex vivo gene transfer strategies (see,e.g., U.S. Pat. No. 5,399,346), which can be expensive and beyond thecost of the general population.

These shortcomings associated with the use of currently availablevectors for genetic therapeutic treatment of AIDS have led researchersto seek out new viral vectors. One such vector is HIV, itself. HIVvectors have been employed for infectivity studies (Page et al., J.Virol., 64, 5270-5276 (1990)) and for the introduction of genes (such assuicide genes) into CD4+ cells, particularly CD4+ HIV-infected cells(see, e.g., Buchschacher et al., Hum. Gener. Ther., 3, 391-397 (1992);Richardson et al., J. Virol., 67, 3997-4005 (1993); Carroll et al., J.Virol, 68, 6047-6051 (1994); and Parolin et al., J. Virol., 68,3888-3895 (1994)). The strategy of these studies is to use HIV vectorsto introduce genes into the CD4+ T-cells and monocytic cells.

To date, however, these vectors are extremely complex. Moreover, use ofthese vectors is accompanied by a risk of generating wild-type HIV viaintracellular recombination. Cotransfection/coinfection of defectivevector sequences and helper virus has been observed to result inrecombination between homologous regions of the viral genomes (Inoue etal., PNAS, 88, 2278-282 (1991)). Observed complementation in vitroindicates that a similar replication-defective HIV vector couldrecombine in vivo, thus exacerbating an already existing HIV infection.The fact that retroviruses package two RNA genomes into one virion hasled researchers to suggest that retroviruses carry two viral RNAs tocircumvent any genetic defects caused by complementation and/orrecombination (Inoue et al. (1991), supra).

In addition to the risk of intracellular recombination, therebyresulting in wild-type HIV, HIV vectors have an associated risk ofmutation in vivo, which increases the pathogenicity of the viral vector.This has lead Sarver et al. (AIDS Res. and Hum. Retrovir., 9, 483-487(1993b)) to speculate regarding the development of second-generationrecombinant HIV vectors, which are replication-competent, yetnonpathogenic. Such vectors, in comparison with the predominantly usednonreplicating vectors (i.e., replication-deficient vectors) continue toreplicate in a patient, thus providing constant competition withwild-type HIV. So far, however, such vectors are not available.

Ideally, the best opportunity to treat an infected individual occurs atthe time of inoculation, before the virus even infects the host.However, this is difficult to accomplish inasmuch as many individuals donot realize they have become infected with HIV until the clinical latentphase of disease. Based on this, the stage at which antiviralintervention is most sorely needed is during clinical latency. Therapyat this stage requires that the challenge presented by the large numberof already infected CD4+ lymphocytes, which harbor viral genomes, beconfronted. This is no trivial challenge, as evidenced by the fact that,to date, HIV remains incurable and is only poorly treatable by currentlyavailable therapies. An effective vaccine is not forthcoming, and,although inhibitors of reverse transcriptase and protease have beenshown to prevent HIV replication in tissue culture, the development ofviral resistance in vivo has led to treatment failure. Thus, HIV genetherapy may have little benefit for the vast majority of HIV-infectedindividuals, predicted to reach more than 40 million by the year 2000.

In view of the above, it is also becoming increasingly important todevelop long-term and persistent immunological responses to certainpathogens, especially viruses, particularly in the context of AIDS andcancer, for example. Live-attenuated (LA) vaccines, usingreplication-competent, but nonpathogenic viruses have been considered(Daniel et al., Science, 258, 1938-1941 (1992); and Desrosiers, AIDSRes. & Human Retrovir., 10, 331-332 (1994)). However, such nonpathogenicviruses, which differ from the corresponding wild-type viruses by adeletion in one or more genes, either (i) cannot elicit a protectiveimmune response because the antigen does not persist (because theLA-virus does not efficiently replicate); or (ii) the LA-virusreplicates but has other pathogenic potential, as witnessed by theability of the LA-virus to cause disease in young animal models (Baba etal., Science, 267, 1823-1825 (1995)).

For the aforementioned reasons, there remains a need for alternativeprophylactic and therapeutic treatment modalities of viral infection,particularly in the context of AIDS and cancer. The present inventionprovides such alternative methods by providing a conditionallyreplicating vector. The invention also provides additional methods inwhich such a vector can be employed. These and other objects andadvantages of the present invention, as well as additional inventivefeatures, will be apparent from the description of the invention setforth herein.

BRIEF SUMMARY OF THE INVENTION

The present invention provides a conditionally replicating viral vector,which is characterized by a capacity to replicate only in a host cellthat is permissive for replication of the vector.

In one embodiment, the conditionally replicating viral vector comprisesat least one nucleic acid sequence, the presence, transcription ortranslation of which confers to the vector in a replication-permissivehost cell a selective advantage over a wild-type strain of viruscorresponding to the virus from which the vector was derived.

In another embodiment of the conditionally replicating viral vector, thevector, which preferably is a retrovirus, comprises at least one nucleicacid sequence, the presence, transcription or translation of whichconfers to a host cell, which is infected with the vector, a selectiveadvantage over a cell infected with a wild-type strain of viruscorresponding to the virus from which the vector was derived.

Also provided by the present invention is a pharmaceutical compositioncomprising a conditionally replicating viral vector and apharmaceutically acceptable carrier. Further provided is a host cellcomprising a conditionally replicating viral vector. A vector, whereinsaid vector, if DNA, comprises a nucleotide sequence selected from thegroup consisting of SEQ ID NOS:2, 3, 4, 5, 6, 14, in which at least oneN is mutated, 15 and 16 and wherein said vector, if RNA, comprises anucleotide sequence encoded by a nucleotide sequence selected from thegroup consisting of SEQ ID NOS:2, 3, 4, 5, 6, 14, in which at least oneN is mutated, 15 and 16 is also provided as are isolated and purifiednucleic acid molecules as set forth herein. Similarly provided are amethod of engendering a vector with a ribozyme, a method of modifying avector, and a method of propagating and selectively packaging aconditionally replicating vector without using a packaging cell line.

In yet another embodiment of the present invention, a method oftherapeutically and prophylactically treating a host cell for a viralinfection is provided. Such methods can additionally comprise the use ofa helper-expression vector, a cytotoxic drug, proteins/factors, or aprotease/reverse transcriptase inhibitor as appropriate. The method canbe used, for example, to inhibit replication of a virus, treat cancer,in vivo gene transfer, or to express a gene of interest in a host cell.

In still yet another embodiment, a method of using a host cellcomprising a conditionally replicating vector to detect interactionbetween a drug/factor and a protein is provided. Such a method enablesprotein characterization and screening of drugs/factors for activitywith respect to a given protein.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1E are schematic depictions of the structure of the viralgenome present in wild-type HIV (FIG. 1A), crHIV-1.1 (FIG. 1B),crHIV-1.11 (FIG. 1C), crHIV-1.12 (FIG. 1D), and crHIV-1.111 (FIG. 1E).Designations: cr, conditionally replicating; U5, U5 coding sequence; Rz,ribozyme; ψ, packaging signal; gag, pol and env, the coding sequence forproteins that form the viral core, reverse transcriptase, and envelope,respectively; tat, rev, rre, and nef, additional viral genes; openboxes, viral long-terminal repeats. The crosses in the wild-type U5coding region indicate the approximate regions in which ribozymesaccording to the invention cleave in the wild-type U5 RNA, but notmodified crHIV U5 RNA (i.e., "mU5").

FIG. 2 depicts the DNA sequences of wild-type HIV U5 RNA SEQ ID NO:1!(A) and modified crHIV U5 RNA SEQ ID NO:2! (B). Numbers refer to thenumber of bases downstream from the start of transcription.

FIG. 3 is a graph depicting reverse transcriptase activity (cpm/μl)versus time (days after co-transfection) for crHIV-mediated inhibitionof wild-type HIV replication in Jurkat cells co-transfected withwild-type HIV and crHIV-1.1 (open boxes), with wild-type HIV andcrHIV-1.11 (open crossed boxes), with wild-type HIV and crHIV-1.12(stippled boxes), and with wild-type HIV and control plasmid pGEM-3Z(solid boxes).

FIG. 4 is a graph depicting reverse transcriptase activity (cpm/μl)versus time (days after co-transfection) for crHIV-mediated inhibitionof wild-type HIV replication in Jurkat cells co-transfected withwild-type HIV and crHIV-1.1 (open boxes), wild-type HIV and crHIV-1.11(open crossed boxes), wild-type HIV and crHIV-1.111 (stippled boxes),and wild-type HIV and plasmid pGEM-3Z (solid boxes).

FIGS. 5A-5C are schematic depictions of the primers and probes employedto detect U5 RNA transcripts from wild-type HIV (FIG. 5A), crHIV-1.1(FIG. 5B), and crHIV-1.111 (FIG. 5C). Designations: U5, U5 codingsequence; ψ, packaging signal; gag, pol and env, the coding sequencesfor proteins that form the viral core, reverse transcriptase, andenvelope, respectively; open boxes, viral long terminal repeats; solidboxes, tat and rev coding sequences; and PE, V1, V2, V3, R1 and R2,primers employed for wild-type and/or conditionally replicating viruses.The cross in the wild-type U5 coding region indicates the approximateregion in which ribozymes according to the invention cleave in thewild-type U5 RNA, but not modified crHIV U5 RNA (i.e., "mU5").

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides a method of inhibiting the replication ofa wild-type strain of a virus. The method comprises contacting a host,which is capable of being infected with such a wild-type strain ofvirus, and preferably is actually infected with such a wild-type strainof virus, with a vector that is propagated only in a host that ispermissive for the replication of the vector (i.e., a nonpathogenic,conditionally replicating (cr) vector).

As further described herein, a particular aim of the method is toestablish a competitive infection in the host with such a nonpathogenic,conditionally replicating vector. Generally, a conditionally replicatingvector according to the invention comprises at least one nucleic acidsequence that confers a selective advantage for replication and spreadto the conditionally replicating vector as compared with a wild-typevirus, and/or at least one nucleic acid sequence that confers aselective advantage for propagation of viral particles to a host cellcontaining a conditionally replicating vector as compared with a hostcell containing a wild-type virus.

In a preferred embodiment of the invention, the vector comprises an HIVsequence and is employed for treatment of HIV infection. Thus, thevector, or a host cell containing the vector, comprises at least onenucleic acid sequence that (1) provides a crHIV genome with a selectiveadvantage over a wild-type HIV genome for packaging into progeny virions(i.e., in cells where they both reside), and/or (2) provides a host cellproducing a conditionally replicating vector (virus) with a selectiveadvantage for production of a crHIV virion, as compared with a host cellproducing a wild-type virus. One method (to which the invention is notlimited) is to confer crHIV genomes with a selective advantage forpackaging by providing them with one or more ribozymes capable ofcleaving the wild-type HIV genome.

Wild-Type Virus

According to the invention, a "virus" is an infectious agent thatconsists of protein and nucleic acid, and that uses a host cell'sgenetic machinery to produce viral products specified by the viralnucleic acid. A "nucleic acid" refers to a polymer of DNA or RNA that issingle or double-stranded, linear or circular, and, optionally, containssynthetic, nonnatural, or modified nucleotides, which are capable ofbeing incorporated into DNA or RNA polymers. A DNA polynucleotidepreferably is comprised of genomic or cDNA sequences.

A "wild-type strain of a virus" is a strain that does not comprise anyof the human-made mutations as described herein, i.e., any virus thatcan be isolated from nature. Alternatively, a wild-type strain is anyvirus that has been cultured in a laboratory, but still, in the absenceof any other virus, is capable of producing progeny genomes or virionslike those isolated from nature. For example, the pNL4-3 HIV molecularclone described in the following Examples is a wild-type strain which isavailable from the AIDS Research and Reference Reagent Program Catalogthrough the National Institutes of Health (see, also, Adachi et al., J.Virol., 59, 284-291 (1986)).

In general, the method of the present invention preferably is employedto treat viral diseases that result from viral infection. Desirably, avirus (as well as the vector, as discussed below) is a RNA virus, butalso can be a DNA virus. RNA viruses are a diverse group that infectsprokaryotes (e.g., the bacteriophages) as well as many eukaryotes,including mammals and, particularly, humans. Most RNA viruses havesingle-stranded RNA as their genetic material, although at least onefamily has double-stranded RNA as the genetic material. The RNA virusesare divided into three main groups: the positive-stranded viruses (i.e.,those of which the genome transferred by the virus is translated intoprotein, and whose deproteinized nucleic acid is sufficient to initiateinfection), the negative-stranded viruses (i.e., those of which thegenome transferred by the virus is complementary to the message sense,and must be transcribed by virion-associated enzymes before translationcan occur), and the double-stranded RNA viruses. The method of thepresent invention preferably is employed to treat positive-strandedviruses, negative-stranded viruses, and double-stranded RNA viruses.

As employed herein, a RNA virus encompasses Sindbis-like viruses (e.g.,Togaviridae, Bromovirus, Cucumovirus, Tobamovirus, Ilarvirus,Tobravirus, and Potexvirus), Picornavirus-like viruses (e.g.,Picornaviridae, Caliciviridae, Comovirus, Nepovirus, and Potyvirus),minus-stranded viruses (e.g., Paramyxoviridae, Rhabdoviridae,Orthomyxoviridae, Bunyaviridae, and Arenaviridae), double-strandedviruses (e.g., Reoviridae and Birnaviridae), Flavivirus-like viruses(e.g., Flaviviridae and Pestivirus), Retrovirus-like viruses (e.g.,Retroviridae), Coronaviridae, and other viral groups including, but notlimited to, Nodaviridae.

A preferred RNA virus according to the invention is a virus of thefamily Flaviviridae, preferably a virus of the genus Filovirus, andespecially a Marburg or Ebola virus. Preferably, a virus of the familyFlaviviridae is a virus of the genus Flavivirus, such as yellow fevervirus, dengue virus, West Nile virus, St. Louis encephalitis virus,Japanese encephalitis virus, Murray Valley encephalitis virus, Rociovirus, tick-borne encephalitis virus, and the like.

Also preferred is a virus of the family Picornaviridae, preferably ahepatitis A virus (HAV), hepatitis B virus (HBV), or a non-A or non-Bhepatitis virus.

Another preferred RNA virus is a virus of the family Retroviridae (i.e.,a retrovirus), particularly a virus of the genus or subfamilyOncovirinae, Spumavirinae, Spumavirus, Lentivirinae, and Lentivirus. ARNA virus of the subfamily Oncovirinae is desirably a humanT-lymphotropic virus type 1 or 2 (i.e., HTLV-1 or HTLV-2) or bovineleukemia virus (BLV), an avian leukosis-sarcoma virus (e.g., Roussarcoma virus (RSV), avian myeloblastosis virus (AMV), avianerythroblastosis virus (AEV), and Rous-associated virus (RAV; RAV-0 toRAV-50), a mammalian C-type virus (e.g., Moloney murine leukemia virus(MuLV), Harvey murine sarcoma virus (HaMSV), Abelson murine leukemiavirus (A-MuLV), AKR-MuLV, feline leukemia virus (FeLV), simian sarcomavirus, reticuloendotheliosis virus (REV), spleen necrosis virus (SNV)),a B-type virus (e.g., mouse mammary tumor virus (MMTV)), and a D-typevirus (e.g., Mason-Pfizer monkey virus (MPMV) and "SAIDS" viruses). ARNA virus of the subfamily Lentivirus is desirably a humanimmunodeficiency virus type 1 or 2 (i.e., HIV-1 or HIV-2, wherein HIV-1was formerly called lymphadenopathy associated virus 3 (HTLV-III) andacquired immune deficiency syndrome (AIDS)-related virus (ARV)), oranother virus related to HIV-1 or HIV-2 that has been identified andassociated with AIDS or AIDS-like disease. The acronym "HIV" or terms"AIDS virus" or "human immunodeficiency virus" are used herein to referto these HIV viruses, and HIV-related and -associated viruses,generically. Moreover, a RNA virus of the subfamily Lentiviruspreferably is a Visna/maedi virus (e.g., such as infect sheep), a felineimmunodeficiency virus (FIV), bovine lentivirus, simian immunodeficiencyvirus (SIV), an equine infectious anemia virus (EIAV), and a caprinearthritis-encephalitis virus (CAEV).

A virus according to the invention also desirably is a DNA virus.Preferably, the DNA virus is an Epstein-Barr virus, an adenovirus, aherpes simplex virus, a papilloma virus, a vaccinia virus, and the like.

Many of these viruses are classified as "Biosafety Level 4" (i.e., WorldHealth Organization (WHO) "Risk Group 4") pathogens for which maximumcontainment facilities are required for all laboratory work. Theordinary skilled artisan, however, is familiar with and is capable ofadhering to the safety precautions necessary for these viruses.

A "host cell" can be any cell, and, preferably, is a eukaryotic cell.Desirably, the host cell is a lymphocyte (such as a T lymphocyte) or amacrophage (such as a monocytic macrophage), or is a precursor to eitherof these cells, such as a hematopoietic stem cell. Preferably, the cellscomprise a CD4+ glycoprotein on the cell surface, i.e., are CD4+.Desirably, however, a CD4+ T lymphocyte, which has been infected withthe AIDS virus, has not yet become activated (i.e., preferablyexpression of nef has not yet occurred, and, even more preferably, CD4gene expression has not been downregulated, as further discussed below).Moreover, a host cell preferably is a cell that lacks the CD4 marker,and yet is capable of being infected by a virus according to the presentinvention. Such a cell includes, but is not limited to, an astrocyte, askin fibroblast, a bowel epithelial cell, and the like. Preferably, thehost cell is of a eukaryotic, multicellular species (e.g., as opposed toa unicellular yeast cell), and, even more preferably, is a mammalian,e.g., human, cell. A cell can be present as a single entity, or can bepart of a larger collection of cells. Such a "larger collection ofcells" can comprise, for instance, a cell culture (either mixed orpure), a tissue (e.g., epithelial or other tissue), an organ (e.g.,heart, lung, liver, gallbladder, urinary bladder, eye, and otherorgans), an organ system (e.g., circulatory system, respiratory system,gastrointestinal system, urinary system, nervous system, integumentarysystem or other organ system), or an organism (e.g., a bird, mammal, orthe like). Preferably, the organs/tissues/cells being targeted are ofthe circulatory system (e.g., including, but not limited to heart, bloodvessels, and blood), respiratory system (e.g., nose, pharynx, larynx,trachea, bronchi, bronchioles, lungs, and the like), gastrointestinalsystem (e.g., including mouth, pharynx, esophagus, stomach, intestines,salivary glands, pancreas, liver, gallbladder, and others), urinarysystem (e.g., such as kidneys, ureters, urinary bladder, urethra, andthe like), nervous system (e.g., including, but not limited to, brainand spinal cord, and special sense organs, such as the eye) andintegumentary system (e.g., skin). Even more preferably, the cells beingtargeted are selected from the group consisting of heart, blood vessel,lung, liver, gallbladder, urinary bladder, and eye cells.

Vector

A "vector" is a nucleic acid molecule (typically DNA or RNA) that servesto transfer a passenger nucleic acid sequence (i.e., DNA or RNA) into ahost cell. Three common types of vectors include plasmids, phages andviruses. Preferably, the vector is a virus.

Desirably, the vector is not a wild-type strain of a virus, inasmuch asit comprises human-made mutations. Thus, the vector typically is derivedfrom a wild-type viral strain by genetic manipulation (i.e., bydeletion) to comprise a conditionally replicating virus, as furtherdescribed herein. Optimally, the viral vector comprises a strain ofvirus that is of the same type as the wild-type virus causing theinfection being treated, which, preferably, is one of the aforementionedwild-type viruses. Accordingly, preferably, the vector is derived from aRNA virus, even more preferably, the vector is derived from aretrovirus, and, optimally, the vector is derived from a humanimmunodeficiency virus. Such a vector derived from a humanimmunodeficiency virus is referred to generically herein as a "crHIV"vector.

A vector also, preferably, is a "chimeric vector," e.g., a combinationof a viral vector with other sequences, such as, for instance, acombination of HIV sequences with another virus (which, desirably, isderived from a wild-type viral strain to comprise a conditionallyreplicating vector). In particular, HIV sequences desirably can belinked with sequences of a modified (i.e., non-wild-type) strain ofadenovirus, adeno-associated virus, a Sindbis virus vector, or anamphotropic murine retroviral vector.

As encompassed herein, a vector can comprise either DNA or RNA. Forinstance, either a DNA or RNA vector can be used to derive the virus.Similarly, a cDNA copy can be made of a viral RNA genome. Alternatively,a cDNA (or viral genomic DNA) moiety can be transcribed in vitro toproduce RNA. These techniques are well-known to those skilled in theart, and also are described in the following Examples.

A "conditionally replicating virus" is a replication-defective virus,which is defective only under certain conditions. In particular, thevirus can complete its replicative cycle in a permissive host cell, andcannot complete its replicative cycle in a restrictive host cell. A"permissive host cell" is a host cell infected with a wild-type strainof virus. Such infection can occur either before or after infection witha conditionally replicating virus according to the invention.Alternatively, a "permissive host cell" is one that encodes wild-typeviral gene products necessary for viral replication. Thus, aconditionally replicating vector according to the invention is a virus(which preferably is the same type of virus as the infection beingtreated) that replicates only upon complementation with a wild-typestrain of virus or when wild-type virus infects cells containingconditionally replicating vector genomes.

In a preferred embodiment, a vector comprises an RNA virus (e.g., aconditionally replicating HIV virus), which is introduced in the form ofDNA. This preferred embodiment provides a replicating HIV-1 (crHIV)vector strategy that affords nonpathogenic crHIV-1 vector genomes with aselective advantage over pathogenic wild-type HIV genomes. Specifically,in cells containing both wild-type HIV and crHIV genomes, crHIV RNAshave a selective advantage for packaging into virions because theycontain, for instance, ribozymes that cleave wild-type RNA, but notcrHIV RNA. Such nonpathogenic crHIVs are able to spread to uninfectedcells that are susceptible to HIV infection (e.g., CD4+ cells) in thepresence of wild-type helper virus. In this manner, selective packagingand spread of crHIV interferes with wild-type HIV replication.

In particular, crHIV genomes are introduced into infected cells oruninfected cells. Infected cells supply the crHIV genome with proteinsrequired for encapsidation and production of progeny virions. crHIVgenomes are introduced into uninfected cells preferably either directlyby transduction (e.g., this can be done, for instance, byliposome-mediated transduction of crHIV DNA, or by using a chimericviral vector), or by infection of crHIV particles that result fromtransfection of wild-type HIV-infected cells. Uninfected cells on theirown do not produce crHIV particles. However, they can becomesuperinfected with wild-type virus, which supplies the proteins requiredfor the further production of crHIV particles. In this sense, aconditionally replicating vector according to the invention alsofunctions as a type of "viral delivery vector" that provides the meansby which multiple rounds of crHIV infection (i.e., in the presence ofconcurrent infection with wild-type HIV) can ensue. Such a vector, whichprovides a source of virus for more than one round of viral replication,contrasts with other currently employed vectors, such as those used withpackaging cell lines, and which provide for only a single round ofreplication.

If desired (e.g., to facilitate use of the vector in vitro), wild-typeviral gene products can be co-supplied to a cell infected with theconditionally replicating vector. Wild-type viral gene products can besupplied not only by co-infection with a wild-type viral strain (or aCDNA or provirus of a RNA virus), but also by supplying them to a cellin the form of their genes subcloned in an expression vector, e.g., ahelper expression vector ("helper"), that is capable of imparting on ahost cell transcription or translation of the sequences (regulatory orstructural), or, alternatively, the gene products can be suppliedexogenously, i.e., by adding the protein products to the cell. Withrespect to the "helper," its expression can be cell specific or notcell-specific and it can be introduced into a host cell in concert witha conditionally replicating viral vector as defined herein and, thereby,enable continuous replication of the conditionally replicating viralvector.

As used herein, "complementation" refers to the nongenetic interactionof viral gene products from different sources in cells. Specifically,with a mixed infection, complementation comprises an enhancement in theviral yield of one or both parental genomes, while the genotypes of theparental genomes remain unchanged. Complementation can be nonallelic(i.e., intergenic, wherein mutants defective in different functionsassist each other in viral replication by supplying the function that isdefective in the other virus) or allellic (i.e., intragenic, wherein thetwo parents have defects in different domains of a multimeric protein).

Desirably, the types of cells that can be transfected (transduced) withcrHIV DNA (i.e., by liposomes or by using an adenoviral vector or anamphotropic retroviral vector) can be either HIV-infected or uninfectedcells. HIV infected cells can be activated or unactivated. If they areactivated, they will immediately transcribe wild-type HIV RNA and crHIVRNA, resulting in selective packaging of crHIV RNA into progeny virions.If HIV-infected cells are not activated, the crHIV DNA will reside inthem until they become activated (e.g., through stimulation by mitogens,antigens, and the like), resulting again in selective packaging of crHIVRNA into progeny virions. Both activated and unactivated uninfectedcells that are transfected with crHIV DNA will not produce virions untilthey become superinfected with wild-type HIV and activated bystimulation, resulting again in selective packaging of crHIV RNA intoprogeny virions.

Superinfection of cells containing crHIV genomes (e.g., as a result oftransfection or infection) occurs because crHIV genomes do not encodeviral proteins that block superinfection (such as env and nef). Theresulting crHIV virions can infect uninfected cells because the viralparticles contain the reverse transcriptase molecule, which all HIVparticles carry so that they can create a DNA provirus from theirgenomic RNA. This process is called reverse transcription. Once crHIVvirions infect uninfected cells, they can undergo reverse transcriptionand produce a provirus from their genomic RNA. Thus, these cells are theequivalent to those uninfected cells that are directly transduced withcrHIV DNA. They cannot produce crHIV particles until these cells becomesuperinfected with wild-type HIV and become activated, then once again,selective packaging of crHIV RNA into progeny virions occurs. It ispossible that crHIV particles could also infect some cells that arealready infected with HIV (see, e.g., Yunoki et al., Arch. Virol., 116,143-158 (1991); Winslow et al., Virol., 196, 849-854 (1993); Chen etal., Nuc. Acids Res., 20, 4581-4589 (1992); and Kim et al., AIDS Res. &Hum. Retrovir., 9, 875-882 (1993)). However, for this to occur, theseHIV-infected cells must not express proteins that down-regulate CD4expression, because this will prevent the crHIV virions from infectingthese cells. Activated, HIV-infected cells generally down-regulate CD4expression. Accordingly, HIV-infected cells that are not activated arepotentially susceptible to crHIV superinfection and, thus, could beanother source for crHIV particle production With a preferred crHIVvector according to the invention, the vector comprises sequencesrequired for RNA transcription, tRNA primer binding, dimerization andpackaging, and either lacks sequences encoding proteins that blocksuperinfection with wild-type HIV (erg., nef or env proteins) orcomprises such sequences but they are either not transcribed or nottranslated into functional protein, such that their expression is deemed"silent." Even more preferably, the vector lacks the region or sequencescoding the region of wild-type HIV from within the gag coding sequenceto and including the nef gene. Optimally, however, the vector doescomprise the rev responsive element (RRE), which is cloned into thevector in the region of the deletion or some other convenient region.Such a preferred HIV vector is said to "lack the region or sequencescoding the region" inasmuch as this vector can be administered in itsRNA manifestation, or, alternatively, as DNA, as previously described.

Vector construction is well-known to those skilled in the art. Forinstance, and as described in Example 1, the DNA manifestation of a RNAvirus, such as HIV, is cleaved using restriction enzymes to excise HIVencoding sequences from within the gag coding region to within the U3region, following the nef gene. A cloning cassette comprised of apolylinker containing multiple restriction sites is inserted into theregion of the deletion prior to ligation to provide convenientrestriction sites for cloning into the vector. A DNA fragment containingRRE is subcloned into one of these sites. The resultant vector producesa truncated gag transcript, and does not produce wild-type Gag protein,or any other wild-type HIV proteins. Moreover, it is not necessary thatthe vector express even the truncated gag protein inasmuch as the gagtranslation initiation sequence can be mutated to prevent itstranslation.

Using the same approach, the crHIV sequences can be linked to othersequences, such as those of a virus or other vector, to derive achimeric vector. For instance, the crHIV sequences can be ligated tothose of Sindbis virus, AAV, adenovirus, or amphotropic retrovirus toname but a few such viruses that can be used to provide for delivery ofthe crHIV sequences. With such a chimeric vector, the vector can beintroduced into the cell either using the conjoined virus's mechanismfor cell entry (e.g., receptor-mediated endocytosis for adenovirus) orother means, e.g., liposomes.

Preferably, according to the invention, a vector (i.e., a conditionallyreplicating virus that preferably is a crHIV vector) comprises at leastone nucleic acid sequence, the possession (i.e., presence, transcriptionor translation) of which confers a selective advantage. There are twotypes of such nucleic acid sequences contemplated for inclusion in thevector: (1) a nucleic acid sequence, the possession of which optimallyconfers a selective advantage for viral replication and spread to avector comprising such a sequence over a wild-type strain of virus(i.e., preferably, a wild-type strain from which the vector was derived,and which does not comprise the sequence), and (2) a nucleic acidsequence, the possession of which optimally confers a selectiveadvantage to cells infected with a vector comprising the sequence ascompared with cells infected with a wild-type strain of virus (i.e.,preferably, a wild-type strain from which the vector was derived (andalso, for example, a helper-expression vector that promotes vectorreplication and/or function in an uninfected host cell), and which doesnot comprise the sequence) by, for example, promoting cell survival,promoting vector particle production and/or propagation, promoting theproduction of crHIV vector virions from crHIV vector-producing cells,inducing apoptosis, facilitating protein production or promotingimmunological function or targeting, so as to achieve a desiredprophylactic, therapeutic or biological outcome. Each of thesesequences, or a plurality of each of these sequences, i.e., a sequencethat alone or in combination with another factor(s), promotes thepropagation of the vector and/or promotes a particular host cellfunction so as to enable a favorable prophylactic, therapeutic and/orbiological outcome, can be included in the vector, either in the absenceor the presence of the other sequence, i.e., the vector can comprise "atleast one nucleic acid sequence" and "at least one additional nucleicacid sequence."

A "nucleic acid" is as previously described. A "nucleic acid sequence"in particular comprises any gene or coding sequence (i.e., DNA or RNA)of potentially any size (i.e., limited, of course, by any packagingconstraints imposed by the vector), the possession of which confers aselective advantage, as further defined herein. A "gene" is any nucleicacid sequence coding for a protein or a nascent mRNA molecule(regardless of whether the sequence is transcribed and/or translated).Whereas a gene comprises coding sequences as well as noncoding sequences(e.g., regulatory sequences), a "coding sequence" does not include anynoncoding DNA.

1. Nucleic acid sequence, the possession of which confers a selectiveadvantage in a host cell to a vector comprising such a sequence over awild-type strain of virus.

A nucleic acid sequence, which confers a selective advantage to a vectorin a host cell over a wild-type strain of virus, preferably is anysequence that allows viral particles propagated from the vector to beselectively produced or packaged as compared with viral particlespropagated from the wild-type virus. Such sequences include, but are notlimited to, a sequence that results in an increase in the number ofvector genomes produced intracellularly as compared with wild-typegenomes, and an antiviral nucleic acid sequence.

The first category of nucleic acid sequences that confer a selectiveadvantage in a host cell to a vector containing the sequence as comparedwith a wild-type strain of virus are sequences such as a promoter A"promoter" is a sequence that directs the binding of RNA polymerase andthereby promotes RNA synthesis, and that can comprise one or moreenhancers. "Enhancers" are cis-acting elements that stimulate or inhibittranscription of adjacent genes. An enhancer that inhibits transcriptionalso is termed a "silencer." Enhancers differ from DNA-binding sites forsequence-specific DNA binding proteins found only in the promoter (whichalso are termed "promoter elements") in that enhancers can function ineither orientation, and over distances of up to several kilobase pairs(kb), even from a position downstream of a transcribed region.

Accordingly, preferably, the promoter (e.g., the long-terminal repeat(LTR)) of a conditionally replicating HIV vector is modified such thatthe vector is more responsive to certain cytokines than is the wild-typeHIV strain. For instance, a modified HIV promoter is available thatdemonstrates increased transcriptional activity in the presence ofinterleukin-2. Incorporation of this promoter into a vector andintroduction of the vector into wild-type, HIV-infected cells preferablyresults in increased production and packaging of progeny virions fromthe vector genome as compared with the wild-type HIV genome. Othercytokines and/or chemokines (e.g., including, but not limited to, tumornecrosis factor α, RANTES, and the like) similarly can be employed topromote selective packaging of virions encoded by the vector.

The second category of a nucleic acid sequence that confers a selectiveadvantage to a vector containing the sequence as compared with awild-type strain of virus includes, as a preferred nucleic acidsequence, an antiviral nucleic acid sequence. "Antiviral agents" arecategorized by their mode of action, e.g., inhibitors of reversetranscriptase, competitors for viral entry into cells, vaccines,protease inhibitors, and genetic antivirals. "Genetic antiviral agents"are DNA or RNA molecules that are transferred into cells and affecttheir intracellular targets either directly (i.e., as introducedintracellularly) or after their conversion to either RNA or protein(reviewed by Dropulic et al. (1994), supra). A genetic antiviralsequence also is a preferred nucleic acid sequence. Genetic antiviralagents include, but are not limited to, antisense molecules, RNA decoys,transdominant mutants, toxins, immunogens, and ribozymes. Desirably, agenetic antiviral is an antisense molecule, an immunogen, and aribozyme. Accordingly, a preferred nucleic acid sequence that confers aselective advantage to a vector over a wild-type strain of virus is thatof a genetic antiviral agent selected from the group consisting of anantisense molecule, an immunogen, and a ribozyme.

An "antisense molecule" is a molecule that mirrors a short segment of agene whose expression is to be blocked. An antisense molecule directedagainst HIV hybridizes to wild-type HIV RNA, allowing its preferentialdegradation by cellular nucleases. Antisense molecules preferably areDNA oligonucleotides, desirably of about 20 to about 200 base pairs inlength, preferably about 20 to about 50 base pairs in length, and,optimally, less than 25 base pairs in length. An antisense molecule canbe expressed from crHIV RNA that preferentially binds to genomicwild-type RNA, thereby providing the crHIV RNA with a selectiveadvantage for packaging into progeny virions.

An "immunogen" is a single-chain antibody (scAb) directed to a viralstructural protein. An immunogen is transferred as nucleic acid andexpressed intracellularly. Similarly, an immunogen also can be anyantigen, surface protein (including those that are class-restricted) orantibody, which facilitates vector and/or host cell selection. In apreferred vector, the nucleic acid sequence comprises a scAb that bindsto wild-type HIV Rev protein. This preferably prevents maturation of Revprotein by resulting in its withholding in the endoplasmic reticulum.Specifically, Rev proteins export unspliced HIV RNA to the cytoplasm bybinding to the RRE and then oligomerizing to surround the HIV RNA. HIVRNAs that are complexed with Rev are exported into the cytoplasm andbypass the cell's splicing machinery. Thus, if wild-type Rev does notbind to the wild-type RRE, then wild-type HIV RNAs are not exported intothe cytoplasm, and are not encapsidated into progeny virions.

Optimally, the vector containing the scAb nucleic acid sequence furthercomprises a modified RRE sequence, and encodes a mutated Rev proteinthat recognizes the modified, but not the wild-type, RRE. Accordingly,in cells containing wild-type HIV and a vector comprising the scAbnucleic acid sequence, the vector preferentially is packaged intovirions. A similar strategy preferably is employed wherein proteins ofthe wild-type HIV matrix or nucleocapsid (i.e., or any protein involvedin protein/RNA interactions that affect encapsidation of viral RNA) arethe targets of the scAb.

A "ribozyme" is an antisense molecule with catalytic activity, i.e.,instead of binding RNA and inhibiting translation, ribozymes bind RNAand effect site-specific cleavage of the bound RNA molecule. Generally,there are four ribozyme groups: the Tetrahymena group I interveningsequence, RNase P, and the hammerhead and hairpin ribozymes. Howeveradditional catalytic motifs also exist in other RNA molecules, e.g.,hepatitis delta virus and ribosomal RNAs in fungal mitochondria.

A preferred ribozyme is a ribozyme in which the catalytic domain cleavesa 3'-nucleotide NUH sequence, wherein N can be any nucleotide (i.e., G,A, U or C), and H can be either an A, C or U. However, inasmuch as thesequence that is cleaved most efficiently by such ribozymes is a GUCsite, preferably the NUH sequence comprises a GUC site.

Desirably, such a ribozyme cleaves in a region of a wild-type strain ofvirus or its transcripts, but does not cleave in a region of a vector orits transcripts. The ribozyme cleaves the virus or its transcripts inthe sense that such a virus or vector can be either RNA or DNA, aspreviously described. By cleavage "in a region" is meant cleavage in atargeted region, i.e., preferably a region of the virus that isnecessary for viral propagation. Desirably, the vector has been modifiedso that this particular region being targeted (i.e., if present in thevector at all) is not cleaved by the ribozyme. Optionally, the ribozymecan cleave the vector, so long as cleavage does not occur in a regionrequired for propagation of viral, e.g., crHIV particles.

Optimally, the ribozyme is encoded by a sequence selected from the groupconsisting of SEQ ID NO:3(i.e.,CACACAACACTGATGAGGCCGAAAGGCCGAAACGGGCACA) and SEQ ID NO:4 (i.e.,ATCTCTAGTCTGATGAGGCCGAAAGGCCGAAACCAGAGTC). Whereas SEQ ID NO:3 comprisesa ribozyme that is targeted to the +115 site (i.e., in terms of thenumber of bases downstream from the start of transcription) of thewild-type HIV U5 region, SEQ ID NO:4 comprises a ribozyme that istargeted to the +133 site of the wild-type HIV U5 region.

Such a ribozyme is able to cleave within the wild-type HIV genome (orits transcripts) but not the vector genome (or its transcripts) inasmuchas the vector U5 sequences are modified by in vitro site-directedmutagenesis, such as is known in the art and described in Example 1. Inparticular, the vector sequences preferably are modified such that thevector comprises a sequence selected from the group consisting of SEQ IDNO:2 (i.e., GTGTGCCCACCTGTTGTGTGACTCTGGCAGCTAGAGAAC), SEQ ID NO:5,(i.e., GTGTGCCCGCCTGTTGTGTGACTCTGGTAACTAGAGATC), SEQ ID NO:6 (i.e.,GTGTGCCCGTCTGTTGTGTGACTCTGGCAAC TAGAGATC), SEQ ID NO:14, in which atleast one N is mutated, SEQ ID NO:15 and SEQ ID NO:16. In the form ofRNA, the vector preferably comprises a sequence encoded by a sequenceselected from the group consisting of SEQ ID NO:5, SEQ ID NO:6, SEQ IDNO:14, in which at least one N is mutated, SEQ ID NO:15 and SEQ IDNO:16. In contrast, wild-type HIV comprises the US sequence encoded bythe sequence of SEQ ID NO:1 (i.e.,GTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATC). The modifications in toto andcomparison to the wild-type U5 sequence (in the form of DNA) are set outin FIG. 2.

Moreover, other ribozymes targeted to other regions of a viral and,particularly, a HIV genome can be employed, either alone or incombination. For instance, the ribozyme can cleave within other RNAsequences needed for viral replication, e.g., within the reversetranscriptase, protease, or transactivator protein, within Rev, orwithin other necessary sequences, such as have been described.Preferably, a vector comprises multiple ribozymes, e.g., targeted tomultiple sites. In such cases, the analogous sequences in the vector aremodified by site-directed mutagenesis, or some other means such as isknown in the art, to derive a vector that is resistant to such ribozymecleavage.

When the vector is a human immunodeficiency virus, preferably the vectorlacks the tat gene and its 5' splice site and, in place thereof,comprises a triple anti-Tat ribozyme cassette, wherein the catalyticdomain of each ribozyme of the triple ribozyme cassette cleaves adifferent site on a wild-type human immunodeficiency viral nucleic acidmolecule, in particular a different site within tat. Preferably, thecatalytic domain of each ribozyme cleaves a nucleotide sequence in aregion of a nucleic acid molecule of wild-type human immunodeficiencyvirus for which there is no ribozyme-sensitive counterpart in thevector, itself.

2. Nucleic acid sequence, the possession of which confers a selectiveadvantage to cells infected with a vector comprising the sequence ascompared with cells infected with a wild-type strain of virus.

A nucleic acid sequence that confers a selective advantage to a cellcontaining a vector comprising the sequence over a cell containing awild-type strain of virus (i.e., that lacks the sequence) preferably isany sequence that allows a cell containing the vector to survive andpropagate viral particles (i.e., crHIV viral particles) as compared witha cell containing the wild-type virus. Such sequences include, but arenot limited to, any sequence that allows the cell, or the vectorcontained in the cell, to escape destruction, sequences that promotecell survival, sequences that induce apoptosis, sequences thatfacilitate protein production or sequences that promote immune functionor targeting.

For instance, preferably such a nucleic acid sequence contained on thevector encodes genes for multidrug resistance (see., e.g., Ueda et al.,Biochem. Biophys. Res. Commun., 141, 956-962 (1986); Ueda et al., J.Biol. Chem., 262, 505-508 (1987); and Ueda et al., PNAS, 84, 3004-3008(1987)). In the presence of added cytotoxic drug (e.g., as used forcancer chemotherapy), this allows a cell containing the vector tosurvive, whereas a cell that contains wild-type virus, such as HIV, doesnot. Such cytotoxic drugs include, but are not limited to, actinomycinD, vinblastine sulfate, vincristine sulfate, daunomycin, adriamycin,VP-16, and AMSA.

Alternatively, such a nucleic acid sequence desirably comprises asequence selected from the group consisting of a sequence of (or asequence that encodes) a mutated (i.e., mutant) protease, and a sequenceof (or a sequence that encodes) a mutated (i.e., mutant) reversetranscriptase. Preferably, a mutated reverse transcriptase is engineeredto be resistant to nucleoside and non-nucleoside reverse transcriptaseinhibitors, and a mutated protease is engineered to be resistant tocommonly employed protease inhibitors.

Administration of these protease or reverse transcriptase inhibitors toa host in conjunction with the vector is employed to select for cellsproducing the vector as opposed to cells producing the wild-type virus.Similarly, this approach is modified for use with any drug that inhibitsviral replication such that the virus can be mutated to escape frominhibition. Accordingly, for treatment of HIV, the selective nucleicacid sequence incorporated into the vector preferably comprises mutatedHIV sequences. Optimally, however, these sequences do not preventsuperinfection with wild-type HIV.

Preferably, the vector is one of those set forth above, and, inparticular, the preferred crHIV vectors depicted in FIGS. 1B-1E, i.e.,crHIV-1.1, crHIV-1.11, crHIV-1.12, and crHIV-1.111, respectively(Dropulic' et al., PNAS, 93, 11103-11108 (1996)). Also preferred is avector as described in Example 11, i.e., cr2HIV.

The cr2HIV vector preferably lacks the tat gene and its splice site fromthe genome of a wild-type human immunodeficiency virus. In place of thetat gene and its splice site, the cr2HIV vector comprises a tripleanti-Tat ribozyme cassette, wherein the catalytic domain of eachribozyme of the triple ribozyme cassette cleaves a different site on awild-type HIV nucleic acid molecule. Preferably, the catalytic domain ofeach ribozyme of the triple ribozyme cassette cleaves a different sitewithin tat on a wild-type HIV nucleic acid molecule. More preferably,the catalytic domain of each ribozyme cleaves a nucleotide sequence in aregion of a nucleic acid molecule of wild-type HIV for which there is noribozyme-resistant counterpart in the vector, itself.

Optimally, a vector is compatible with the cell into which it isintroduced, e.g., is capable of imparting expression on the cell of thevector-encoded nucleic acid sequences. Desirably, the vector comprisesan origin of replication functional in the cell. When a nucleic acidsequence is transferred in the form of its DNA coding sequence (e.g.,versus in the form of a complete gene comprising its own promoter),optimally the vector also contains a promoter that is capable of drivingexpression of the coding sequence and that is operably linked to thecoding sequence. A coding sequence is "operably linked" to a promoter(e.g., when both the coding sequence and the promoter togetherconstitute a native or recombinant gene) when the promoter is capable ofdirecting transcription of the coding sequence.

In a recombinant vector of the present invention, preferably all theproper transcription (e.g., initiation and termination signals),translation (e.g., ribosome entry or binding site and the like) andprocessing signals (e.g., splice donor or acceptor sites, if necessary,and polyadenylation signals) are arranged correctly on the vector, suchthat any gene or coding sequence is appropriately transcribed (and/ortranslated, if so desired) in the cells into which the vector isintroduced. The manipulation of such signals to ensure appropriateexpression in host cells is well within the knowledge and expertise ofthe ordinary skilled artisan.

Preferably, the vector also comprises some means by which the vector orits contained subcloned sequence is identified and selected. Vectoridentification and/or selection is accomplished using a variety ofapproaches known to those skilled in the art. For instance, vectorscontaining particular genes or coding sequences preferably areidentified by hybridization, the presence or absence of so-called"marker" gene functions encoded by marker genes present on the vectors,and/or the expression of particular sequences. In the first approach,the presence of a particular sequence in a vector is detected byhybridization (e.g., by DNA-DNA hybridization) using probes comprisingsequences that are homologous to the relevant sequence. In the secondapproach, the recombinant vector/host system is identified and selectedbased upon the presence or absence of certain marker gene functions suchas resistance to antibiotics, thymidine kinase activity, and the like,caused by particular genes encoding these functions present on thevector. In the third approach, vectors are identified by assaying for aparticular gene product encoded by the vector. Such assays are based onthe physical, immunological, or functional properties of the geneproduct.

Accordingly, the present invention also provides a vector, which, ifDNA, comprises a nucleotide sequence selected from the group consistingof SEQ ID NOS:2, 4, 5, 6, 7, 15, 16, 17 and 18 and, which, if RNA,comprises a nucleotide sequence encoded by a nucleotide sequenceselected from the group consisting of SEQ ID NOS:4, 5, 6, 7.

The present invention further provides a method of engendering a vector,which is derived from a wild-type human immunodeficiency virus and whichis capable of replicating only in a host cell that is permissive forreplication of said vector, with a ribozyme. The ribozyme, which iscomprised within or encoded by the vector, cleaves a nucleic acid of awild-type human immunodeficiency virus but not the vector, itself, andits transcripts, if any. The method comprises obtaining a vector, whichis derived from a wild-type human immunodeficiency virus and which iscapable of replicating only in a host cell that is permissive forreplication of said vector, and incorporating into the vector a nucleicacid sequence, which comprises or encodes a ribozyme, the catalyticdomain of which cleaves a nucleic acid of a wild-type humanimmunodeficiency virus but not the vector, itself, and its transcripts,if any. In such a method, the nucleotide sequence comprising or encodingthe U5 sequence of the wild-type human immunodeficiency virus can bedeleted from the vector and replaced with a nucleotide sequence selectedfrom the group consisting of SEQ ID NOS:2, 5, 6, 14, in which at leastone N is mutated, 15 and 16 if the vector is DNA, and a nucleotidesequence encoded by a nucleotide sequence selected from the groupconsisting of SEQ ID NOS:2, 5, 6, 14, in which at least one N ismutated, 15 and 16, if the vector is RNA. Preferably, the vectorreplicates in a host cell permissive for replication of said vector morethan once.

Also provided by the present invention is a method of modifying avector. The method comprises obtaining a vector and introducing into thevector a nucleotide sequence selected from the group consisting of theDNA sequences of SEQ ID NOS:2, 2, 3, 4, 5, 6, 14, in which at least oneN is mutated, 15 and 16, if the vector is DNA, and a nucleotide sequenceencoded by a nucleotide sequence selected from the group consisting ofSEQ ID NOS:2, 4, 5, 6, 7, 15, 16, 17 and 18, if the vector is RNA.

Further provided by the present invention is a method of propagating andselectively packaging a conditionally replicating vector without using apackaging cell line. The method comprises contacting the conditionallyreplicating vector with a cell capable of being infected by anothervector, which is the same type of vector as the conditionallyreplicating vector and which differs from the conditionally replicatingvector by being wild-type for replication competency; subsequentlycontacting the cell with the other vector; and then culturing the cellunder conditions conducive to the propagation of the conditionallyreplicating vector.

Also provided is an isolated and purified nucleic acid molecule selectedfrom the group consisting of a DNA molecule comprising a nucleotidesequence selected from the group consisting of SEQ ID NOS:2, 5, 6, 14,in which at least one N is mutated, 15 and 16 and a RNA moleculecomprising a nucleotide sequence encoded by a nucleotide sequenceselected from the group consisting of SEQ ID NOS:2, 6, 7, 15, 16, 17 and18.

Method of Use

The above-described vectors preferably are introduced into a host cellfor the prophylactic and therapeutic treatment of viral infection, forease of vector maintenance, as well as for other reasons. Accordingly,the present invention provides a host cell comprising a vector accordingto the invention. The isolation of host cells, and/or the maintenance ofsuch cells or cell lines derived therefrom in culture, has become aroutine matter, and one in which the ordinary skilled artisan iswell-versed.

In particular, a vector as described above preferably is employed in theprophylactic and therapeutic treatment of a viral infection, preferablysuch as where the infection is from a wild-type virus, preferably awild-type RNA virus, even more preferably, from a wild-type retrovirus,and optimally from a wild-type HIV.

The method comprises contacting a host cell, which is capable of beinginfected with a wild-type virus, with a conditionally replicatingvector, which is capable of being replicated only in a host cellpermissive for the replication of the vector, the presence,transcription or translation of which inhibits the replication of thewild-type strain of virus in the host cell. Desirably, the vectorreplicates more than once and comprises at least one nucleic acidsequence, the possession (i.e., presence, transcription or translation)of which confers a selective advantage in a host cell to the vector overa wild-type strain of virus, which, optimally, is the strain from whichthe vector was derived.

According to this method, the nucleic acid sequence preferably comprisesa nucleotide sequence, which comprises or encodes a genetic antiviralagent, which adversely affects the replication and/or expression of avirus other than said vector. Desirably, the genetic antiviral agent isselected from the group consisting of an antisense molecule, a ribozyme,and an immunogen. Optimally, the genetic antiviral agent is a ribozyme,preferably the catalytic domain of which cleaves at a 3' nucleotide NUHsequence (i.e., especially a GUC sequence). Optionally, the ribozyme isencoded, at least in part, by a sequence selected from the groupconsisting of SEQ ID NO:3 and SEQ ID NO:4. Desirably, the ribozymecleaves in a region of the wild-type strain of virus or its transcripts,but does not cleave in a region of the vector or its transcripts.Preferably, this is because the wild-type strain of virus comprises asequence encoded by SEQ ID NO:1, whereas the vector, if DNA, comprises anucleotide sequence selected from the group consisting of SEQ ID NOS:2,5, 6, 14, in which at least one N is mutated, 15 and 16, and, if RNA,comprises a nucleotide sequence encoded by a nucleotide sequenceselected from the group consisting of SEQ ID NOS:2, 5, 6, 14, in whichat least one N is mutated, 15 and 16.

The method also desirably is carried out wherein the vector comprises atleast one nucleic acid sequence, the possession (i.e., presence,transcription or translation) of which confers a selective advantage toa host cell infected with the vector over a cell infected with awild-type strain of virus, which, optimally, is the strain of virus fromwhich the vector was derived. In this regard, a vector can comprise atleast one nucleic acid sequence, which confers a selective advantage toa host cell infected with the virus and at least nucleic acid sequence,which confers a selective advantage to the vector over a wild-typestrain of a virus corresponding to the virus from which the vector wasderived.

Accordingly, the method preferably is carried out wherein the nucleicacid sequence comprises a nucleotide sequence encoding a multidrugresistance. Alternatively, the method is carried out wherein the nucleicacid sequence comprises a nucleotide sequence encoding a mutated(mutant) protease and a nucleotide sequence encoding a mutated (mutant)reverse transcriptase, such as when the viral infection to beprophylactically or therapeutically treated is a retrovirus.

The method preferably further comprises administering to a host cell anagent selected from the group consisting of a cytotoxic drug, a proteaseinhibitor, and a reverse transcriptase inhibitor (i.e., in addition toadministration of the vector).

Accordingly, a vector can be employed in accordance with theabove-described method not only to treat therapeutically a viralinfection but to protect a potential host cell from viral infection,i.e., a method of prophylactically treating a viral infection or a"vaccination" against a virus of interest, such as a RNA virus, inparticular a retrovirus, such as HIV. The method essentially inhibitsthe replication of a wild-type strain of virus before the host cellcomes into contact with the wild-type strain of virus. In this regard,the vector can comprise or encode proteins that block superinfectionwith a wild-type virus. The method comprises contacting the host cellwith a conditionally replicating vector, as described above, and a"helper-expression vector," i.e., a viral genome that promotes thereplication of the "vector" in an uninfected host. The conditionallyreplicating vector comprises a selective advantage for packaging and/orpropagation. Furthermore, the vector, for example, can contain asequence that enhances cell survival, promotes viral production, inducesapoptosis, facilitates protein production and/or promotes immunefunction and/or targeting. The "helper-expression vector" construct isany expression vector that complements for the inability of the "vector"to replicate. Such helper-expression vectors are common and are easilyconstructed by those of ordinary skill in the art. The helper-expressionvector can be either packaged into virions, like the vector, orexpressed without a packaging requirement. Since the "vector" has aselective advantage for packaging and/or propagation, this systemprovides a safe means to achieve high replication of the virus withoutthe possible pathogenic effects that a live attenuated virus couldpotentially cause. In addition, the vector can be admixed withnonspecific adjuvants to increase immunogenicity. Such adjuvants areknown to those skilled in the art, and include, but are not limited toFreund's complete or incomplete adjuvant, emulsions comprised ofbacterial and mycobacterial cell wall components, and the like.

When a vector is employed in accordance with the above-described methodas a prophylactic treatment of viral infection, the vector can encode anantigen of a protein that is not encoded by a wild-type virus, such as amutant viral protein or a nonviral protein. Accordingly, the antigenencoded by the vector can be of bacterial origin or cancerous cellorigin, for example. Furthermore, the "vector" also can encode a MHCgene for proper presentation of the antigen to the host's immune system.Thus, such vectors can be used to facilitate a persistent immunologicalresponse against a diverse array of potential pathogens and/orendogenous proteins (e.g., tumor-specific antigen) that are selectivelyexpressed in abnormal cells.

Furthermore, the "helper-virus" (also referred to herein as "helper")expression vector can be engineered to express only in specific celltypes (e.g., stem cells, professional antigen presenting cells, andtumor cells) by the addition or omission of a specific geneticelement/factor (either in the vector or helper-virus expressionconstruct), which permits cell-specific vector replication and spread.Thus, the vector still spreads by complementation with the helper-virusconstruct, but this spread is cell-specific, depending upon whether acertain genetic element/factor is added to or omitted from the vector orhelper-virus expression construct. This can be used alone or incombination with other of the above-mentioned strategies.

For example, a conditionally replicating HIV vector can be designed toreplicate specifically in macrophages, rather than in T-cells. Thevector, which would constitute a Tat-defective HIV (the vector encodesthe other HIV proteins but they are not expressed because of the absenceof the Tat transcriptional transactivator), can encode a ribozyme thatcleaves wild-type HIV but not conditionally replicating HIV RNA. Thehelper-expression vector for this vector can encode a tat gene expressedoff of a macrophage-specific promoter. Thus, the crHIV wouldconditionally replicate only in macrophage cells, while not being ableto replicate in T-cells or other cell types.

Alternatively, the tat gene can be operably linked to a tumor-specificpromoter; thus, the crHIV would then replicate only in tumor CD4 cellsand not in normal CD4 cells. The genetic element/factor also can be amodification of a promoter sequence of the vector such that it isexpressed only in specific cell types and not in other cell types inconcert with the "helper-virus" expression construct.

In another embodiment, the helper-expression construct or the vectorconstruct envelope proteins (if such constructs are engineered tocontain envelope proteins) can be modified so that the vector-virionwill specifically infect certain cell types (e.g., tumor cells), whilenot being able to infect other cell types (e.g., normal cells). In yetanother embodiment, an adenovirus, which is lacking one or several keyfactors for replication, could be complemented by using a helperconstruct, which provides such factors linked to a tumor-specificpromoter. Thus, the factors that complement replication of theadenovirus would only be expressed in tumor cells, thereby permittingviral replication in tumor cells (with expression of proteins requiredfor cell killing), but not in normal cells.

Thus, the present invention also provides a method of treating cancer,and in particular, treating T-cell leukemia. "Treating cancer" accordingto the invention comprises administering to a host a further modifiedvector as set forth herein for the purpose of effecting a therapeuticresponse. Such a response can be assessed, for example, by monitoringthe attenuation of tumor growth and/or tumor regression. "Tumor growth"includes an increase in tumor size and/or the number of tumors. "Tumorregression" includes a reduction in tumor mass.

"Cancer" according to the invention includes cancers that arecharacterized by abnormal cellular proliferation and the absence ofcontact inhibition, which can be evidenced by tumor formation. The termencompasses cancer localized in tumors, as well as cancer not localizedin tumors, such as, for instance, those cancer cells that expand from atumor locally by invasion, or systemically by metastasis. Theoretically,any type of cancer can be targeted for treatment according to theinvention. Preferably, however, the cancer is of viral origin.

Finally, the above-described vectors can be directly used for in vivogene therapy. Current strategies for gene therapy suffer because theycannot mediate gene delivery to large percentage of cells; only acertain percentage of the cells are infected. This is especiallyimportant in anti-tumor strategies where gene transduction of the entiretumor population is crucial. By adding the "vector" in concert with a"helper," the immediately transduced cells will produce viral particlesthat can infect neighboring cells and thus enable high and possiblecomplete transduction efficiency. In one embodiment to this invention, ahuman retrovirus (which could be HIV or a retrotransposon element) couldbe delivered into tissue (or cells in vitro) with a "helper" construct.Cells immediately containing the vector and helper will produce virusand will package the vector conditionally into virions. These virionswill be able to mediate high efficiency transduction of neighboringcells (since cell-cell contact is the most efficient means to transducecells) The immediately transduced cells may or may not die, dependingwhether the vector/helper combination results in a cytolytic infection.In the case of a retrotransposon, the helper may not need to containstructural proteins since normal or tumor cells may contain theprotein/factor necessary for encapsidation into virions. In this casethe helper can merely be, but not restricted to, a transactivatorprotein that activates transcription of the factors required forretrotransposon encapsidation. In the case of HIV, other factors may,but not necessarily, be required for encapsidation of the HIV genomeinto progeny virions for infection/transduction of cells.

The above-described vectors also can be used in counter-biological andcounter-chemical warfare strategies. For example, a conditionallyreplicating vector can be delivered into an individual recently infectedwith a highly pathogenic virus or bacterium or a chemical agent (e.g.,toxin). The vector would interfere with the replication of thepathogenic virus as described previously. However, the conditionallyreplicating vector also can be used for antibacterial or anti-chemicalstrategies in concert with a helper-expression vector ("helper").

For example, a conditionally replicating vector can secreteanti-bacterial or anti-toxin antibodies after a "helper" permits itsexpression and propagation. The "helper" can be, but not necessarily,driven off an inducible promoter that permits its expression uponactivation by a bacterium, a cytokine (in response to bacterialinfection), an antibiotic (as with the tetracycline inducible promotersystems (Paulus et al., J. Virol., 70, 62-67 (1996)) or a chemical(e.g., the toxin, itself). Thus, the conditionally replicating vectorwould not only selectively propagate with the aid of the "helper" inresponse to the incurring pathogen or toxin (as a result of activationof the helper) but also secrete anti-pathogen or anti-toxin antibodiesto inhibit the pathological effects of the tumor antigen, pathogen orchemical (e.g., toxin). Thus, any protein, factor, or genetic elementthat can be transcribed into either mRNA and/or protein can be insertedinto a conditionally replicating vector to inhibit a pathogenicresponse--in concert with a "helper," which promotes its selectivepropagation and expression (selective because the products of the helperare expressed conditionally (for example, but not restricted to, (a) aninducible promoter system--a factor in a tumor cell activates theproduction of a helper factor, a toxin responsive sequence thatexpresses a helper factor, or a cytokine responsive promoter inducesproduction of a helper factor, (b) a helper RNA/protein/factor isselectively stabilized in certain cells and not in others), and (c)indirect induction of a third party gene that affects helper viralprotein production, chaperoning, targeting, structure or anotherbiofunction). Such strategies can be used in transgenic plants andanimals to protect them from pathogens. Similarly, such strategies canbe used in transgenic systems to produce heterologous proteins/factorsof value.

In another embodiment of a method in accordance with the presentinvention, a cell line can be developed for screening a drug/factor todetermine, for example, which part of the protein/factor is importantfor a particular function. A vector can be created to express amutagenized protein of interest within a given cell line. The RNAencoding the mutagenized protein, however, is made resistant to theribozyme by insertion of silent point mutations, for example. Wild-typeprotein expression, however, is inhibited within the cell line. Vectorsthat express a ribozyme to the protein of interest also can beconstructed to express mutant test protein. When the vector istransduced into the cells, most of the native RNA encoding the normalprotein is cleaved, whereas the mutant test protein is expressed. Thismethod can be used with recently developed delivery and selectiontechniques as a quick and powerful technique to determine how a givenprotein functions and how a given factor/drug interacts with theprotein.

There also are numerous uses of the method and the vectors of thepresent invention in vitro. For instance, the vectors can be employed toascertain certain nuances of viral replication and ribozyme function.Similarly, the ribozyme-containing vectors can be used as diagnostictools, e.g., to assess mutations present in diseased cells, or toexamine genetic drift. This aforementioned discussion is by no meanscomprehensive regarding the use of the present invention.

Benefits of the Invention

The advantages of using a crHIV strategy for genetic therapeutictreatment of AIDS and other viruses are considerable. For instance, theproblem of targeting the vector to cells infected by HIV becomesresolved. After in vivo transfection of crHIVs into infected CD4+ cells,the crHIVs become packaged into progeny virions using the endogenousinfectious HIV envelope proteins. Thus, the crHIV RNA tags along insideprogeny virions and infects cell types that are normally infectable bythat particular strain of HIV, producing nonpathogenic virions. Thisincludes difficult to target cells, such as the microglia of the brain,which are a major reservoir for HIV infection of the central nervoussystem. There is likely to be little toxicity associated with crHIVvectors that infect uninfected CD4+ cells, since no viral proteins arecoded by crHIV vectors. Moreover, the result of crHIV vector competitionwith wild-type HIV results in the production of nonpathogenic particles,which results in decreased viral loads. Decreasing pathogenic HIV-1loads can not only increase the survival time of infected individuals,but also can decrease the rate of transmission to uninfectedindividuals, since the crHIV particles also can spread systemically(i.e., as does infectious HIV). Decreased pathogenic HIV-1 loads in theblood can be particularly important in pregnant HIV-infectedindividuals, since the production of crHIVs can also decreasetransmission of HIV-1 from infected mothers to their fetuses in utero.

The plasmid DNA/lipid mixture that can be employed for introducing thecrHIV vector into host cells should be stable and cheap to produce,bypassing expensive ex vivo strategies. Of course, the method of theinvention is inherently flexible inasmuch as it could also be employedfor ex vivo gene delivery, should this be desired. Regardless, theavailability of the liposome-mediated approach opens the possibility fortreatment of the general population--something that is not feasible withcurrent gene therapeutic strategies. The crHIV vectors also can beengineered to contain several ribozymes, which can be made to differenttargets on the HIV genome. This reduces the possibility that infectiousHIV can mutate and escape the effect of the anti-HIV ribozymes.Furthermore, the conditionally replication competent virus strategy canbe applied to treat other viral infections, especially those where viralturnover is high.

A particularly useful feature of crHIV vectors is that they can beemployed to express genetic antiviral agents, for instance, a ribozyme,post-transcriptionally. Thus, infection of uninfected cells with crHIVvectors results in low toxicity because little expression occurs fromthe HIV long-terminal repeat (LTR) promoter in the absence of the Tatprotein. High levels of crHIV expression, and its consequent antiviralactivity, occurs only when the Tat protein is provided bycomplementation with wild-type HIV. Thus, crHIV vectors are not designedto protect cells from HIV infection, but to lower the overall wild-typeHIV viral burden through selective accumulation of nonpathogenic crHIVparticles.

While not seeking to be bound by any particular theory regarding theoperation or functioning of the invention, it is believed that ribozymescan be employed as confirmed in the following Examples to provide crHIVgenomes with a selective advantage because of two useful properties: (1)they have a high degree of specificity, and (2) they have a relativeefficiency, depending upon their ability to co-localize with target RNAs(Cech, Science, 236, 1532-1539 (1987)). The specificity of ribozymes isconferred by their specific hybridization to complementary targetsequences containing a XUY site. Ribozymes are relatively efficientbecause they cleave target RNAs with high efficacy only when theyefficiently co-localize with target RNAs. In a mixed HIV/crHIVinfection, co-localization of ribozyme-containing crHIV RNAs withwild-type HIV RNAs must occur, since HIV RNA genomes dimerize prior topackaging into progeny virions. Cleavage of non-genomic species ofwild-type HIV RNAs, required for the production of viral proteins, islikely to be less efficient than that of genomic wild-type HIV RNAsinasmuch as non-genomic HIV RNAs no not dimerize. It was discovered inthe experiments described herein that the selective advantage conferredto crHIV RNAs was due to the selective packaging of crHIVs into viralparticles. These results suggest that most efficient cleavage occursintracellularly during dimerization, resulting in the selectivedestruction of wild-type HIV RNAs by host nucleases. This allows for thepreferential packaging of crHIV RNAs into viral particles.

The application of crHIV vectors for HIV therapy can involve not onlygenomic selection of crHIVs, but also cellular selection of cellsproducing crHIV particles. Otherwise, the cells producing wild-type HIVswill produce wild-type HIV particles at a selective advantage over thecells producing crHIV particles, and will rapidly predominate. Aselective advantage can be conferred to crHIV expressing cells byinserting a gene into crHIV genomes (e.g., the multidrug resistancegene) that confer crHIV expressing cells (in the presence of drug) witha survival advantage over cells expressing wild-type HIV. Under theseconditions, wild-type HIV-expressing cells progressively die, but stillproduce some wild-type HIV, while crHIV-expressing cells thatselectively produce crHIV survive. Infection of crHIV-containing cellswith remaining wild-type HIV will result in the further production ofcrHIV containing viral particles. Thus, a viral genomic shift can resultwith the cumulative infection of CD4+ cells with crHIV genomes, therebyaltering the viral balance in the host from pathogenic wild-type HIV tononpathogenic crHIV genomes. Such a strategy can result in clearance ofwild-type HIV, once the balance of HIV genomes selectively shifts fromwild-type HIV to crHIV. Viral replication eventually ceases, sincecrHIVs can only replicate in the presence of wild-type HIV helpergenomes. Therefore, under such mutually restrictive conditions, it canbe possible to engineer crHIV vectors that not only decrease wild-typeHIV viral loads, but also clear the virus from the HIV-infected host.

Means of Administration

According to the invention, a vector is introduced into a host cell inneed of gene therapy for viral infection as previously described. Themeans of introduction comprises contacting a host capable of beinginfected with a virus with a vector according to the invention.Preferably, such contacting comprises any means by which the vector isintroduced into a host cell; the method is not dependent on anyparticular means of introduction and is not to be so construed. Means ofintroduction are well-known to those skilled in the art, and also areexemplified herein.

Accordingly, introduction can be effected, for instance, either in vitro(e.g., in an ex vivo type method of gene therapy) or in vivo, whichincludes the use of electroporation, transformation, transduction,conjugation or triparental mating, transfection, infection, membranefusion with cationic lipids, high-velocity bombardment with DNA-coatedmicroprojectiles, incubation with calcium phosphate-DNA precipitate,direct microinjection into single cells, and the like. Other methodsalso are available and are known to those skilled in the art.

Preferably, however, the vectors or ribozymes are introduced by means ofcationic lipids, e.g., liposomes. Such liposomes are commerciallyavailable (e.g., Lipofectin®, Lipofectamine™, and the like, supplied byLife Technologies, Gibco BRL, Gaithersburg, Md.). Moreover, liposomeshaving increased transfer capacity and/or reduced toxicity in vivo(e.g., as reviewed in PCT patent application no. WO 95/21259) can beemployed in the present invention. For liposome administration, therecommendations identified in the PCT patent application no. WO 93/23569can be followed. Generally, with such administration the formulation istaken up by the majority of lymphocytes within 8 hr at 37° C., withinmore than 50% of the injected dose being detected in the spleen an hourafter intravenous administration. Similarly, other delivery vehiclesinclude hydrogels and controlled-release polymers.

The form of the vector introduced into a host cell can vary, dependingin part on whether the vector is being introduced in vitro or in vivo.For instance, the nucleic acid can be closed circular, nicked, orlinearized, depending on whether the vector is to be maintainedextragenomically (i.e., as an autonomously replicating vector),integrated as a provirus or prophage, transiently transfected,transiently infected as with use of a replication-deficient orconditionally replicating virus, or stably introduced into the hostgenome through double or single crossover recombination events.

Prior to introduction into a host, a vector of the present invention canbe formulated into various compositions for use in therapeutic andprophylactic treatment methods. In particular, the vector can be madeinto a pharmaceutical composition by combination with appropriatepharmaceutically acceptable carriers or diluents, and can be formulatedto be appropriate for either human or veterinary applications.

Thus, a composition for use in the method of the present invention cancomprise one or more of the aforementioned vectors, preferably incombination with a pharmaceutically acceptable carrier. Pharmaceuticallyacceptable carriers are well-known to those skilled in the art, as aresuitable methods of administration. The choice of carrier will bedetermined, in part, by the particular vector, as well as by theparticular method used to administer the composition. One skilled in theart will also appreciate that various routes of administering acomposition are available, and, although more than one route can be usedfor administration, a particular route can provide a more immediate andmore effective reaction than another route. Accordingly, there are awide variety of suitable formulations of the composition of the presentinvention.

A composition comprised of a vector of the present invention, alone orin combination with other antiviral compounds, can be made into aformulation suitable for parenteral administration, preferablyintraperitoneal administration. Such a formulation can include aqueousand nonaqueous, isotonic sterile injection solutions, which can containantioxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and nonaqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The formulations can be presented in unit dose or multidose sealedcontainers, such as ampules and vials, and can be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid carrier, for example, water, for injections, immediatelyprior to use. Extemporaneously injectable solutions and suspensions canbe prepared from sterile powders, granules, and tablets, as describedherein.

A formulation suitable for oral administration can consist of liquidsolutions, such as an effective amount of the compound dissolved indiluents, such as water, saline, or fruit juice; capsules, sachets ortablets, each containing a predetermined amount of the activeingredient, as solid or granules; solutions or suspensions in an aqueousliquid; and oil-in-water emulsions or water-in-oil emulsions. Tabletforms can include one or more of lactose, mannitol, corn starch, potatostarch, microcrystalline cellulose, acacia, gelatin, colloidal silicondioxide, croscarmellose sodium, talc, magnesium stearate, stearic acid,and other excipients, colorants, diluents, buffering agents, moisteningagents, preservatives, flavoring agents, and pharmacologicallycompatible carriers.

An aerosol formulation suitable for administration via inhalation alsocan be made. The aerosol formulation can be placed into a pressurizedacceptable propellant, such as dichlorodifluoromethane, propane,nitrogen, and the like.

Similarly, a formulation suitable for oral administration can includelozenge forms, that can comprise the active ingredient in a flavor,usually sucrose and acacia or tragacanth; pastilles comprising theactive ingredient in an inert base, such as gelatin and glycerin, orsucrose and acacia; and mouthwashes comprising the active ingredient ina suitable liquid carrier; as well as creams, emulsions, gels, and thelike containing, in addition to the active ingredient, such carriers asare known in the art.

A formulation suitable for topical application can be in the form ofcreams, ointments, or lotions.

A formulation for rectal administration can be presented as asuppository with a suitable base comprising, for example, cocoa butteror a salicylate. A formulation suitable for vaginal administration canbe presented as a pessary, tampon, cream, gel, paste, foam, or sprayformula containing, in addition to the active ingredient, such carriersas are known in the art to be appropriate. Similarly, the activeingredient can be combined with a lubricant as a coating on a condom.

The dose administered to an animal, particularly a human, in the contextof the present invention should be sufficient to effect a therapeuticresponse in the infected individual over a reasonable time frame. Thedose will be determined by the potency of the particular vector employedfor treatment, the severity of the disease state, as well as the bodyweight and age of the infected individual. The size of the dose alsowill be determined by the existence of any adverse side effects that canaccompany the use of the particular vector employed. It is alwaysdesirable, whenever possible, to keep adverse side effects to a minimum.

The dosage can be in unit dosage form, such as a tablet or capsule. Theterm "unit dosage form" as used herein refers to physically discreteunits suitable as unitary dosages for human and animal subjects, eachunit containing a predetermined quantity of a vector, alone or incombination with other antiviral agents, calculated in an amountsufficient to produce the desired effect in association with apharmaceutically acceptable diluent, carrier, or vehicle. Thespecifications for the unit dosage forms of the present invention dependon the particular compound or compounds employed and the effect to beachieved, as well as the pharmacodynamics associated with each compoundin the host. The dose administered should be an "antiviral effectiveamount" or an amount necessary to achieve an "effective level" in theindividual patient.

Since the "effective level" is used as the preferred endpoint fordosing, the actual dose and schedule can vary, depending oninterindividual differences in pharmacokinetics, drug distribution, andmetabolism. The "effective level" can be defined, for example, as theblood or tissue level desired in the patient that corresponds to aconcentration of one or more vector(s) according to the invention, whichinhibits a virus, such as HIV, in an assay predictive for clinicalantiviral activity of chemical compounds. The "effective level" forcompounds of the present invention also can vary when the compositionsof the present invention are used in combination with zidovudine orother known antiviral compounds or combinations thereof.

One skilled in the art can easily determine the appropriate dose,schedule, and method of administration for the exact formulation of thecomposition being used, in order to achieve the desired "effectivelevel" in the individual patient. One skilled in the art also canreadily determine and use an appropriate indicator of the "effectivelevel" of the compounds of the present invention by a direct (e.g.,analytical chemical analysis) or indirect (e.g., with surrogateindicators of viral infection, such as p24 or reverse transcriptase fortreatment of AIDS or AIDS-like disease) analysis of appropriate patientsamples (e.g., blood and/or tissues).

Further, with respect to determining the effective level in a patientfor treatment of AIDS or AIDS-like disease, in particular, suitableanimal models are available and have been widely implemented forevaluating the in vivo efficacy against HIV of various gene therapyprotocols (Sarver et al. (1993b), supra). These models include mice,monkeys and cats. Even though these animals are not naturallysusceptible to HIV disease, chimeric mice models (e.g., SCID, bg/nu/xid,bone marrow-ablated BALB/c) reconstituted with human peripheral bloodmononuclear cells (PBMCs), lymph nodes, or fetal liver/thymus tissuescan be infected with HIV, and employed as models for HIV pathogenesisand gene therapy. Similarly, the simian immune deficiency virus(SIV)/monkey model can be employed, as can the feline immune deficiencyvirus (FIV)/cat model.

Generally, an amount of vector sufficient to achieve a tissueconcentration of the administered ribozyme (or vector) of from about 50to about 300 mg/kg of body weight per day is preferred, especially offrom about 100 to about 200 mg/kg of body weight per day. In certainapplications, e.g., topical, ocular or vaginal applications, multipledaily doses are preferred. Moreover, the number of doses will varydepending on the means of delivery and the particular vectoradministered.

In the treatment of some virally infected individuals, it can bedesirable to utilize a "mega-dosing" regimen, wherein a large dose of avector is administered, time is allowed for the compound to act, andthen a suitable reagent is administered to the individual to inactivatethe active compound(s). In the method of the present invention, thetreatment (i.e., the replication of the vector in competition with thevirus being treated) is necessarily limited. In other words, as thelevel, for instance, of HIV decreases, the level of vector dependent onHIV for production of virions will also decrease.

The pharmaceutical composition can contain other pharmaceuticals, inconjunction with a vector according to the invention, when used totherapeutically treat AIDS. These other pharmaceuticals can be used intheir traditional fashion (i.e., as agents to treat HIV infection), aswell as more particularly, in the method of selecting for crHIV virusesin vivo. Such selection as described herein will promote conditionallyreplicating HIV spread, and allow conditionally replicating HIV to moreeffectively compete with wild-type HIV, which will necessarily limitwild-type HIV pathogenicity. In particular, it is contemplated that anantiretroviral agent be employed, such as, preferably, zidovudine.Further representative examples of these additional pharmaceuticals thatcan be used in addition to those previously described, include antiviralcompounds, immunomodulators, immunostimulants, antibiotics, and otheragents and treatment regimes (including those recognized as alternativemedicine) that can be employed to treat AIDS. Antiviral compoundsinclude, but are not limited to, ddI, ddC, gancylclovir, fluorinateddideoxynucleotides, nonnucleoside analog compounds such as nevirapine(Shih et al., PNAS, 88, 9878-9882 (1991)), TIBO derivatives such asR82913 (White et al., Antiviral Research, 16, 257-266 (1991)), andBI-RJ-70 (Shih et al., Am. J. Med., 90(Suppl. 4A), 8S-17S (1991)).Immunomodulators and immunostimulants include, but are not limited to,various interleukins, CD4, cytokines, antibody preparations, bloodtransfusions, and cell transfusions. Antibiotics include, but are notlimited to, antifungal agents, antibacterial agents, andanti-Pneumocystis carinii agents.

Administration of the virus-inhibiting compound with otheranti-retroviral agents and particularly with known RT inhibitors, suchas ddC, zidovudine, ddI, ddA, or other inhibitors that act against otherHIV proteins, such as anti-TAT agents, will generally inhibit most orall replicative stages of the viral life cycle. The dosages of ddC andzidovudine used in AIDS or ARC patients have been published. Avirustatic range of ddC is generally between 0.05 μM to 1.0 μM. A rangeof about 0.005-0.25 mg/kg body weight is virustatic in most patients.The dose ranges for oral administration are somewhat broader, forexample 0.001 to 0.25 mg/kg given in one or more doses at intervals of2, 4, 6, 8, and 12; etc., hr. Preferably, 0.01 mg/kg body weight ddC isgiven every 8 hr. When given in combined therapy, the other antiviralcompound, for example, can be given at the same time as a vectoraccording to the invention, or the dosing can be staggered as desired.The vector also can be combined in a composition. Doses of each can beless, when used in combination, than when either is used alone.

EXAMPLES

The present inventive compounds and methods are further described in thecontext of the following examples. These examples serve to illustratefurther the present invention and are not intended to limit the scope ofthe invention.

Example 1

This example describes the construction of conditionally replicationcompetent vectors according to the invention. In particular, thisexample describes the construction of conditionally replicating vectorsbased on HIV, i.e., crHIV vectors.

One of the most prominent aspects of HIV-1 pathogenesis is theproduction of genetic variants of the virus. The rapid production of HIVvariants in vivo indicates that the virus can be considered within theframework of Darwinian genetic modeling (see, e.g., Coffin, Curr. Top.Microbiol. Immunol., 176, 143-164 (1992); and Coffin, Science, 267,483-489 (1995)). The variants are a result of the infidelity of theHIV-1 reverse transcriptase molecule, which creates mutations in newlytranscribed proviruses from viral genomic RNA. Therefore, under in vivoconditions of no significant bottlenecks and many replicative cycles, asubstantial degree of genetic variation occurs with the production ofmany viral variants. Yet, wild-type HIV still predominates, since, undersuch unrestricted conditions, it has the highest selective advantage.However, in the presence of an inhibitor, for instance zidovudine, aviral variant will be selected that is conferred with a higher selectiveadvantage than the wild-type strain, and consequently will predominate(Coffin (1992) and (1995), supra). Based on this, the present inventionprovides a conditionally replicating viral vector strategy that affordsnonpathogenic HIV-1 genomes with a selective advantage over pathogenicwild-type HIV-1.

These nonpathogenic, conditionally replicating HIV (crHIV) vectors aredefective HIVs that undergo replication and packaging only in cells thatare infected with wild-type HIV. crHIV genomes compete with and decreasepathogenic wild-type HIV viral loads. The effect of decreasing wild-typeHIV viral loads in an infected host should lead to an increased lifeexpectancy. It should also decrease the ability of infected hosts totransmit wild-type HIV to uninfected individuals. For successfulcompetition of crHIVs with wild-type HIV-1, two factors appearimportant: (1) a selective advantage of crHIV genomes over wild-type HIVgenomes, and (2) a selective advantage of crHIV-expressing cells overcells expressing wild-type HIV (i.e., a selective advantage for theproduction of crHIV virions from crHIV-expressing cells over cellsexpressing wild-type HIV).

The crHIV vectors conditionally replicate due to the fact that theycontain the sequences required for RNA expression, dimerization andpackaging, but do not express functional (i.e., wild-type) HIV-1proteins. A selective advantage was imparted to the crHIV vectors byinserting a ribozyme cassette that cleaves in the U5 region of thewild-type HIV genome, but not the crHIV U5 RNA.

The ribozymes present in the vectors do not cleave the crHIV RNA becausethe U5 region of the crHIV RNA has been modified by conserved basesubstitution (base substitutions present in other HIV strains) toprevent the ribozymes from efficiently binding and cleaving these sites.Moreover, the crHIVs are nonpathogenic because they do not code forproteins believed to be responsible for CD4+ cell death. When theHIV-infected cells (that have been transfected with the crHIV vector)become activated, the cells become capable of complementing the crHIVgenomic deficits, resulting in the production of crHIV progeny virions.

In general, crHIV genomes were constructed from the full-length,infectious HIV clone, pNL4-3 (Adachi et al. (1986), supra. All cloningreactions and DNA, RNA, and protein manipulations were carried out usingmethods well known to the ordinary skilled artisan, and which have beendescribed in the art, e.g., Maniatis et al., Molecular Cloning: ALaboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory, NY (1982)).Enzymes and reagents employed in these reactions were obtained fromcommercial suppliers (e.g., New England Biolabs, Inc., Beverly, Mass.;Clontech, Palo Alto, Calif.; and Boehringer Mannheim, Inc.,Indianapolis, Ind.) and were used according to the manufacturers'recommendations. Moreover, vector maintenance and propagation were doneusing techniques that are commonly known, and that have been describedpreviously (e.g., Dropulic' et al. (1992), supra; and Dropulic' et al.(1993), supra).

pNL4-3 was cleaved with the enzymes Pst I (which cleaves in gag, atabout position +1000 from the start of transcription) and Xho I (whichcleaves in nef, at about position +8400 from the start oftranscription), and a polylinker containing convenient restriction siteswas inserted. A 0.86 kb Bgl II to Bam HI fragment containing the revresponsive element (RRE) was cloned into a Bam HI site present in thepolylinker. These manipulations resulted in deletion of the HIVwild-type genome from within the gag coding region to within the U3coding region (i.e., thus also deleting the nef gene). While the vectoris able to produce a truncated gag transcript, a full-length functionalGag protein is not produced by the vector. However, inasmuch aswild-type Gag functions are unnecessary according to the invention, thegag sequences can be mutated to prevent Gag protein from beingtranslated.

A ribozyme cassette containing either single or multiple ribozymes asdescribed herein was inserted into a Sal I site downstream from the BamHI site. To accomplish this, complementary deoxyoligonucleotidesencoding ribozyme sequences were synthesized, annealed and then clonedinto the Sal I site. The ribozymes employed for construction of thecrHIV vectors were hammerhead ribozymes. These ribozymes contained acatalytic domain comprised of 22 base pairs, and two hybridizationdomains comprised of 9 base pairs each. The ribozymes were targetedeither to the +115 or +133 site (i.e., corresponding to the number ofbase pairs downstream from the start of transcription) of the U5 RNAsequence. The hybridization domains and catalytic domain (underlined) ofthe ribozymes targeted to the +115 site and the +133 site are asfollows:

CACACAACACTGATGAGGCCGAAAGGCCGAAACGGGCACA ("the +115 ribozyme") SEQ IDNO:3!

ATCTCTAGTCTGATGAGGCCGAAAGGCCGAAACCAGAGTC ("the +133 ribozyme") SEQ IDNO:4!

The ribozyme cassette was comprised of either a single, double or tripleribozyme(s) placed in tandem. Vectors containing either single (i.e.,"crHIV-1.1" vector, FIG. 1B) or triple (i.e. "crHIV-1.111" vector, FIG.1E) ribozymes were targeted to the same site of the U5 HIV RNA, atposition +115. Vectors containing double ribozymes were targeted eitherto the same site at position +115 (i.e., "crHIV-1.11" vector, FIG. 1C),or to different sites at positions +115 and +133 of the U5 HIV RNA;crHIV-1.12 (i.e., "crHIV-1.12" vector, FIG. 1D). These vectors arereferred to herein generically as "crHIV" vectors.

To complete the construction of the vectors, the crHIV vectors wererendered resistant to ribozyme cleavage (i.e., in their manifestation asRNA) by mutating a site recognized by the hammerhead ribozymes occurringwithin the U5 region of the crHIV genome. To accomplish this, adouble-stranded oligonucleotide (i.e.,AAGCTTGCCTTGAGTGCTCAAAGTAGTGTGTGCCCACCTGTTGTGTGACTCTGGCAGCTAGAGATCCCACAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTGGCGCC SEQ ID NO:13!)containing the base substitutions depicted in FIG. 2 SEQ ID NO:2! wasused to introduce modified sites into the vector. Specifically, basesubstitutions were engineered into the ribozyme hybridization andcleavage sites at base pairs 115 and 133. In particular, as illustratedin FIG. 2; mutations were introduced at base pairs 113, 114, 132, 134and 142. These sites can be modified to comprise any mutation (i.e.,GTGTGCCCNNCTGTTGTGTGACTCTGGNANCTAGAGANC, wherein N can be any mutantnucleotide SEQ ID NO:14!). Preferably, however, the sequences aremutated such that there is, for instance, a G to A substitution at site+113 (i.e., such that the sequence comprisesGTGTGCCCATCTGTTGTGTGACTCTGGTAACTAGAGATC SEQ ID NO:15!), a U (i.e., T, interms of the DNA sequence) to C substitution at site +114 SEQ ID NO:5!,a U (i.e., T, in terms of the DNA sequence) to C substitution at site+132 SEQ ID NO:6!, an A to G substitution at site +134 (i.e., such thatthe sequence comprises GTGTGCCCGTCTGTTGTGTGACTCTGGTAGCTAGAGATC SEQ IDNO:16!) and a U (i.e., T, in terms of the DNA sequence) to Asubstitution at site +142, which mutations can be made either alone, orin combination. In particular, in the absence of other U5 mutations, theU (i.e. T, in terms of the DNA sequence) to C substitution at site +114SEQ ID NO:5! and/or site +132 SEQ ID NO:6! in the crHIV US RNA preventsits scission by ribozymes (Uhlenbeck (1987), supra). The insertedbase-substitutions are present in various other strains of HIV (Myers etal., HIV Sequence Database, Los Alamos Nat. Lab. (1994)), whichindicates that these substitutions do not decrease the replicativecapacity of the HIV genome.

The method as set forth herein can be employed to construct otherconditionally replicating vectors, for instance, comprised of differingviral genomes (e.g., different RNA viruses), or comprised of differentgenetic antiviral agents. Furthermore, a conditionally replicatingvector can be further modified to impart to a host cell, into which thevector is introduced, a selective advantage over a host cell containingthe wild-type virus. For instance, such a vector can be modified tofurther encode multidrug resistance, or a mutated protease or reversetranscriptase.

Example 2

This example describes the resistance to ribozyme cleavage ofconditionally replicating vectors, and, in particular, of the crHIVvectors.

To confirm the resistance to ribozyme cleavage of the crHIV vectors, invitro transcription was performed. To accomplish this, the ribozymesequences were cloned into the Xho I site of pBluescript KSII(Stratagene, La Jolla, Calif.). A 0.21 kilobase pair (kb) Bgl IIfragment containing the mutated crHIV U5 region similarly was excisedfrom the crHIV vector and inserted into the Bam HI site of pBluescriptKSII. The resultant modified pBluescript KSII vectors were linearizedwith Bss HII prior to in vitro transcription. A similar plasmidexpressing wild-type HIV U5 RNA (described in Myers et al. (1994),supra) was employed as a control. It was linearized with Eco RI prior toin vitro transcription.

Radiolabeled U5 HIV RNA and ribozyme RNA were produced by in vitrotranscription of the vectors, as previously described (Dropulic' et al.(1992), supra). The radiolabeled transcripts were incubated together (ata target to ribozyme molar ratio of 1:2) in 1X transcription buffercontaining 40 mM Tris-HCl, pH 7.5, 6 mM MgCl₂, 2 mM Spermidine, and 10mM NaCl. The samples were heated to 65° C., and then cooled to 37° C.for 5 min prior to the addition of stop buffer solution containing 95%formamide, 20 mM EDTA, 0.05% Bromophenol Blue, and 0.05% Xylene CyanolFF. The products were then resolved by denaturing polyacrylamide gelelectrophoresis (PAGE), and detected by autoradiography.

When wild-type U5-HIV RNA was incubated with a transcript containing asingle ribozyme to site +115, cleavage was readily observed. Suchcleavage results in products PI and P2. Cleavage also can be seen whenwild-type HIV RNA was incubated with RNAs containing double ribozymes toeither the same site, or to different sites. When a ribozyme-containingtranscript directed to two different sites was incubated with wild-typeHIV RNA, products P1, P2 and P3 were produced. P3 results from cleavageat the +133 site.

In comparison, when the modified U5-containing crHIV RNA was incubatedwith either a single ribozyme directed to the +115 site, or doubleribozyme directed to either the +115 site or the +133 site, cleavageproducts were not detected. Thus, these results confirm that crHIV U5RNAs are resistant to ribozyme cleavage, while wild-type HIV-U5 RNAs arecleaved by anti-US ribozymes. Moreover, the results validate that theapproach of the present invention can be employed to impartconditionally replicating vectors (including vectors other than crHIVvectors) with a selective advantage for replication when introduced intoa host cell as compared with a wild-type strain of virus.

Example 3

This example describes the ability of ribozyme-containing conditionallyreplicating vectors to cleave wild-type viral RNA intracellularly. Inparticular, this example describes the ability of crHIV vectors tocleave wild-type HIV RNA intracellularly.

The effectiveness of crHIV vector-mediated inhibition of wild-type HIVwas tested by co-transfecting the genomes into Jurkat cells.Transfection was carried out by washing about 106 Jurkat cells in0pti-MEM medium (Life Technologies, Gibco BRL, Gaithersburg, Md.) andthen co-transfecting the cells with about 0.6 μg of wild-type HIV DNA(i.e., pNL4-3) and about 1.8 μg of crHIV DNA. A molar ratio of wild-typeHIV to crHIV provirus of about 1:3 was used to ensure that all cellstransfected with wild-type HIV also contained crHIV proviruses. DNA wasmixed in lipofectin solution (Life Technologies) for 30 min, and thenwas incubated with Jurkat cells for about 3 to about 6 hr, after whichcomplete RPMI 1640 medium containing 10% fetal bovine serum (FBS) wasadded. Virus-containing supernatants were harvested every 2 to 4 days,and virus levels were assayed by reverse transcriptase activity in cellsupernatants, as previously described (Dropulic' et al. (1992), supra).

The effect of crHIV genomes on wild-type HIV replication is shown inFIG. 3. When wild-type HIV was co-transfected with crHIV-1.1, viralgrowth was delayed (FIG. 3, open boxes) relative to cells co-transfectedwith wild-type HIV and a control virus (FIG. 3, closed boxes), but wasnot inhibited. Since anti-U5 ribozymes can inhibit HIV replication invivo under co-localized conditions (e.g., Dropulic' et al. (1992),supra), the viral growth seen could be the result of either: (a)preferential packaging for wild-type HIV RNAs into progeny virions, (b)the production of wild-type HIV RNAs that are resistant to ribozymecleavage, or (c) an accumulation of nonfunctional ribozymes in crHIVRNAs.

The nature of "escape" viral growth was tested by co-transfectingwild-type HIV with crHIV vectors that contain double ribozymes. Ifpreferential packaging of wild-type HIV is responsible for viral growth,then cultures containing double ribozyme crHIVs should have similargrowth kinetics as cultures containing single ribozyme crHIVs. If,however, viral growth results from wild-type HIV RNAs that have becomeresistant to ribozyme action (i.e., as a result of viral reversetranscriptase infidelity), then the kinetics of viral growth should showa greater delay for cultures containing crHIV-1.12 (i.e., directedagainst two viral sites) as compared with cultures containing crHIV-1.11(i.e., directed against a single viral site). Alternatively, if a delayin viral growth was seen that was comparable in cultures containing thedifferent double ribozyme-containing crHIVs, this would suggest that aproportion of the singly expressed ribozymes are nonfunctional in vivo.

As can be seen in FIG. 3, cultures containing crHIV-1.11 (FIG. 3, opencrossed boxes) or crHIV-1.12 (FIG. 3, stippled boxes) showed a greaterdelay in the onset of viral growth than crHIV-1.1, which contained asingly transcribed ribozyme (FIG. 3, open boxes). However, the delay inthe onset of viral growth between crHIV-1.11 and crHIV-1.12 was similar,indicating the correctness of the third possibility, i.e., that singlytranscribed ribozymes are kinetically less efficient in cleaving targetRNAs than are double ribozymes. This suggests that a certain proportionof intracellularly transcribed ribozymes can form in a nonfunctional,possibly misfolded, conformation, since the co-transfection experimentswere performed in a molar excess of ribozyme-containing crHIV genomes.

The ability of multiple ribozymes to relieve this kinetic limitation byproviding a greater probability for functional ribozymes to associatewith wild-type HIV RNAs was explored. For these experiments, Jurkatcells. were co-transfected with wild-type HIV and crHIV-1.111, whichcontains a triple ribozyme to site +115. As can be seen in FIG. 3(stippled boxes), there is no evidence of viral growth with use of atriple ribozyme, even after 22 days in culture. These results areparticularly significant in view of the fact that normal primary T cellsoften die shortly (e.g., about a week) after infection with HIV.

Moreover, these results confirm that ribozyme-containing conditionallyreplicating vectors, such as the crHIV vectors, and particularly thosethat contain multiple ribozymes, can be employed to competeintracellularly with a wild-type viral genome, such as HIV.

Example 4

This example describes an investigation of the mechanism underlying theability of ribozyme-containing conditionally replicating vectors,particularly crHIV vectors, to cleave wild-type viral RNAintracellularly.

For these experiments, cell supernatant RNA from wild-type HIV andcrHIV-1.111 co-transfected cultures was examined with use of the reversetranscription polymerase chain reaction (RT-PCR), as described hereinRT-PCR was done using the primers depicted in FIGS. 5A-C. Namely,ribozyme RNA was detected using primers R1 and R2, wild-type HIV RNA wasdetected using primers V1 and V3, and crHIV RNA was detected usingprimers V2 and V3. Primers R1 (TGTGACGTCGACCACACAACACTGATG SEQ ID NO:7!)and R2 (TGTGACGTCGACTCTAGATGTGCCCGTTTCGGC SEQ ID NO:8! each comprise aSal I restriction site, and amplify the anti-U5 ribozyme RNA by bindingto the ribozyme hybridization sequences. In crHIV-1.111 expressingcells, single, double and triple ribozyme amplification products areseen. Primers VI (GGTTAAGCTTGAATTAGCCCTTCCAGTCCCC SEQ ID NO:9!) and V2(GGTTGGATCCGGGTGGCAAGTGGTCAAAAAG SEQ ID NO:10!) each comprise Bam HI orHin dIII restriction sites, and amplify wild-type HIV RNAs. Along withthe aforementioned V1 primer, the V3(CGGATCCACGCGTGTCGACGAGCTCCCATGGTGATCAG SEQ ID NO:11!) primer comprisesBam HI and other restriction sites. This primer set amplifies crHIV RNAsfrom a crHIV-specific polylinker sequence.

To perform RT-PCR, virion and intracellular RNAs were isolated usingTriZol™ (Life Technologies). Intracellular viral RNAs were isolateddirectly from microcentifuged cell pellets Virion RNAs were isolatedfrom culture supernatants that were first cleared of cells and debris bymicrocentrifugation at 12,000×g for 5 min. Trizol™ was added to thecell-free supernatants, and the mixtures were incubated for 5 min priorto the addition of chloroform for phase separation. The aqueous phasewas transferred to a fresh tube, and the RNA was precipitated withisopropanol using glycogen. After reconstitution of the RNA pellet, theviral RNAs were reverse-transcribed and then amplified by PCR usingradiolabeled primers.

Reverse transcription was performed for 1 hr at 42° C. in first-strandbuffer containing 50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl₂, 5 mMDTT, 1 mM dNTPs, and 20 units (U) of RNase inhibitor, to which 25 U ofMuLV Reverse Transcriptase was added. After reverse transcription wascompleted, the reverse transcriptase was heat-inactivated at 65° C. for10 min. The entire mixture was then added directly to PCR buffer tocomprise a mixture containing a final concentration of 10 rnM Tris-HCl,pH 8.3, 50 mM KCl, and 1.5 mM MgCl₂. The mixture was amplified for 30cycles using 2.5 U of Taq enzyme. The radiolabeled PCR products werethen resolved by denaturing PAGE, and detected by autoradiography.

crHIV-1.111 ribozyme RNAs exist in the supernatants of cells more thantwenty days after co-transfection with wild-type HIV-1 and crHIV-1.111proviral genomes. This is evidenced by the existence of single, doubleand triple ribozyme RNA PCR products. In comparison, no such productsare seen in virions produced by control wild-type, HIV-transfectedcultures. During this period, the cells appeared normal, with noapparent signs of crHIV-induced cytotoxicity. This confirms that crHIVsare packaged into viral particles even though no reverse transcriptaseactivity was observed. Moreover, this indicates that crHIVs can becomplemented intracellularly by HIV gene functions.

Thus, these results indicate that crHIVs inhibit wild-type HIVreplication by inhibiting wild-type HIV spread. The results furtherindicate that other conditionally replicating vectors, for instance,other viral vectors, and/or vectors containing other genetic antiviralscan similarly be employed to inhibit wild-type viral replication andspread.

Example 5

This example describes a further exploration of the mechanism underlyingthe ability of ribozyme-containing, conditionally replicating vectors,particularly crHIV vectors, to cleave wild-type, viral RNAintracellularly.

One possible mechanism is that both wild-type HIV and crHIV RNAs arepackaged into progeny virions, and efficient cleavage occurs in thissmall viral volume due to co-localization of ribozyme and target RNAs.Alternatively, selective packaging of crHIV RNAs into progeny virionscan occur because cleavage of wild-type HIV RNAs predominantly occursintracellularly, and not in the HIV virion. These mechanisms wereexplored herein.

The means by which crHIV-1.111 inhibited wild-type HIV spread wasexamined by RT-PCR of virion- and cell-associated viral RNAs, in cellcultures transfected with wild-type HIV alone, or co-transfected withwild-type HIV and crHIV-1.111. crHIV-1.111 RNAs were exclusively presentin progeny virions produced following co-transfection. In comparison,control, wild-type, HIV-transfected cultures produced virions thatcontained only wild-type HIV RNAs. Intracellularly, both wild-type HIVand crHIV-1.111 RNAs were evident in co-transfected cultures. Therefore,although both wild-type HIV and crHIV RNAs are synthesizedintracellularly, crHIV RNAs are selectively packaged into progenyvirions. This suggests that crHIV-1.111 inhibited wild-type HIV spreadby selectively cleaving genomic wild-type HIV RNAs prior toencapsidation, while allowing some sub-genomic wild-type RNAs to betranslated into proteins for virion production.

To test whether genomic wild-type RNAs are selectively cleaved by crHIVRNAs, the types of intracellular RNAs present in Jurkat cell culturesobtained about 20 days following co-transfection was examined byNorthern hybridization. The probe employed for the Northern blotanalysis, as indicated in FIG. 5, was isolated from a 0.21 kb Bgl IIfragment from the U5 region of pNL4-3.

Cultures transfected with wild-type HIV express all wild-type HIV RNAspecies, i.e., genomic and subgenomic RNA species (FIG. 9, lane 1). Incomparison, crHIV-1.111 co-transfected cultures do not expresssignificant amounts of genomic (9.7 kb), wild-type HIV RNA. RNAs of lowmolecular weight (reflecting the presence of subgenomic wild-type HIVRNAs) were observed in co-transfected cultures. The HIV-RNA smearing inthese samples suggests that some degraded genomic HIV RNAs may bepresent within these low molecular-weight RNAs. In comparison, thesmearing of wild-type HIV RNA from control, wild-type HIV cells is dueto RNA degradation that occurs from the significant CPE observed at thelate stage of HIV infection.

Accordingly, these results confirm that genomic, wild-type HIV RNAs areselectively cleaved and degraded in cells containing wild-type HIV andcrHIV-1.111 genomes, allowing selective crHIV RNA packaging intovirions. Furthermore, these results indicate that the method maysimilarly be employed with other viruses, particularly with other RNAviruses.

Example 6

This example describes an investigation of the ability ofribozyme-containing, conditionally replicating vectors, particularlycrHIV vectors, to undergo the complete viral replicative cycle in thepresence of wild-type helper virus.

To confirm that crHIV genomes undergo the complete viral replicativecycle in the presence of a helper wild-type HIV genome, the productionof virus particles containing crHIV genomes was examined under severalconditions Specifically, first the production of viral particlescontaining crHIV genomes was examined in activated ACH2 cells (AIDSReagent Reference Program, Rockville, Md.) These cells comprise alatently HIV-1 infected cell line. Next, the ability of any crHIVparticles derived from these cultures to infect uninfected Jurkat cellsand produce crHIV DNA was examined.

For these experiments, about 10⁶ ACH2 cells were transfected with about2.5 μg of vector DNA. The cells were stimulated with 50 nM12-O-tetradecanoylphorbol 13-acetate (TPA) about 24 hr aftertransfection. RNA was isolated from the cell supernatants about 72 hrafter transfection. RT-PCR was performed using the R1 and R2 primers asdescribed in Example 4. crHIV ribozyme RNAs were detected in virionsproduced by activated ACH2 cells after transfection with crHIV-1.11, butnot after transfection with pGEM 3Z control plasmid (Promega, Madison,Wis.). Therefore, transfection of crHIV vectors into infected CD4+ cellsresults in the production of viral particles that contain crHIV RNAs.

The ability of crHIV virions derived from these cultures to infectuninfected Jurkat cells and produce crHIV proviruses was examined next.Such proviruses were detected by isolating cellular DNA using Trizol™,cleaving the DNA with Eco RI, and then amplifying ribozymal DNA by PCR,using the R1 & R2 primers as described in Example 4. crHIV DNA wasproduced in Jurkat cells after infection of cell supernatants derivedfrom crHIV-transfected ACH2 cells. Namely, in this case, specificamplification of crHIV-1.11 ribozymal DNA was seen. In comparison, cellsinfected with stimulated ACH2 cell supernatants alone (i.e., in theabsence of any infection of ACH2 cells with crHIV-1.111) showed noribozymal DNA products.

Since crHIV vectors spread only in the presence of wild-type helper HIVgenomes, the ability of uninfected cells containing crHIV genomes to berescued after infection with wild-type HIV was examined. Theseexperiments were carried out by first transfecting cells with crHIV-1.11(i.e., as representative of the crHIV vectors), and then superinfectingwith wild-type HIV (i.e., pNL4-3). Accordingly, about 10⁶ Jurkat cellswere transfected with about 2.5 μg of crHIV DNA. The cells were allowedto grow for about 72 hr prior to infection with wild-type HIV stockvirus. crHIV-1.11 transfected Jurkat cells were incubated with stockpNL4-3 (2×10⁵ TCID₅₀ units per 10⁶ cells) for about 2 hr at 37° C.,washed three times in Opti-MEM® I Reduced Serum Medium, and thenresuspended in complete medium (RPMI 1640 with 10% FBS). RNA wasisolated from cell supernatants as described in Example 4 about 5 daysafter infection.

For the TCID₅₀ assay, supernatants containing HIV were plated out on96-well plates by 5-fold limiting dilution. About 10⁴ MT4 cells (AIDSReagent Reference Program, Rockville, Md.; and Harada et al., Science,229, 563-566 (1985)) were then added to the diluted viral suspensionsand the resultant suspensions were incubated for 7 days until completeviral growth had occurred. MT4 cells are modified T-cells that containthe Tax gene from HTLV-1, which is a transactivator gene that isanalogous to Tat in HIV-1. Supernatants were then assayed for reversetranscriptase activity and scored as previously described (Dropulic' etal. (1992), supra). The tissue culture infectious dose (TCID₅₀) wasdetermined by the method of Reed and Muench (In: Tech. in HIV Res.,Johnson et al., eds., Stockton Press, 71-76 (1990)).

Superinfection of crHIV-transfected Jurkat cells with wild-type HIVresulted in crHIV genomes being rescued into viral particles. The crHIVgenomes are packaged into viral particles after superinfection withwild-type HIV. During this period, the cells appeared normal, with nosignificant signs of cytotoxicity.

These results confirm that crHIV genomes are able to undergo the fullreplicative cycle after complementation with wild-type HIV helper virus.These results also confirm that other viral genomes are likely able toundergo the full replicative cycle after complementation with thecorresponding wild-type virus.

Example 7

This example describes the nature of escape viral growth reported in theprior examples.

The nature of escape viral growth from cultures transfected withwild-type HIV, or co-transfected with wild-type HIV and crHIV-1.11, wasexamined by analyzing virion RNAs using RT-PCR as previously described.Viruses produced by cultures at the early stages of viral growth (i.e.,wild-type HIV transfected culture at day +11, crHIV-1.11 co-transfectedculture at day +19) contained predominantly crHIV RNAs. In comparison,cultures from the late stages of various growth (i.e., wild-type HIVtransfected culture at day +17, crHIV-1.11 co-transfected culture at day+23) contained predominately wild-type HIV RNAs. Therefore, viral growthfrom cells co-transfected with wild-type HIV and crHIV-1.11 provirusesappeared to result from the growth of wild-type HIV that escaped fromintracellular ribozyme restriction. Significantly, crHIV genomes stillcomprised a substantial proportion of the total HIV genomes even incultures at the late stages of viral growth. This suggests that,although wild-type HIV genomes predominated, crHIV genomes were,nevertheless, spreading through the culture, albeit at lowerefficiencies than wild-type HIV genomes.

This confirms that the crHIV vectors, as well as further conditionallyreplicating vectors, can effectively compete with wild-type viralgenomes for viral replication.

Example 8

This example further describes the nature of escape viral growthreported in the prior examples.

The effect of crHIV RNA packaging into virions during escape viralgrowth was studied by measuring infectious wild-type HIV titers.Limiting dilution TCID₅₀ assays (as described in Example 6) wereperformed on viral supernatants from cultures at the exponential stageof viral growth (i.e., wild-type HIV cultures at day +14, crHIV-1.1cultures at day +16, crHIV-1.11 or crHIV-1.12 cultures at day +20). Thesamples were normalized prior to assay using reverse transcriptaseactivity. Supernatants from wild-type HIV, crHIV-1.1, crHIV-1.11 andcrHIV-1.12 cultures had an infectious dose of 1.3×10⁴ TCID₅₀ /ml,5.4×10³ TCID₅₀ /ml, 3.8×10³ TCID₅₀ /ml, and 3.8×10³ TCID₅₀ /ml,respectively. Thus, the packaging of crHIV RNAs into virions duringescape viral growth results in a decrease in the number of infectiouswild-type HIV particles that are produced.

Next examined was whether the decrease in infectious wild-type HIV titerwas the result of cleavage of wild-type HIV RNAs within escaped virions.RNA cleavage products from virions present in the supernatants ofco-transfected cells were assessed by primer extension. The PE primer(GGTTAAGCTTGTCGCCGCCCCTCGCCTCTTG SEQ ID NO:12!) identified in FIG. 6,and which comprises a Hin dIII restriction site, was employed. Primerextension across the cleavage site was performed for 2 hr at 42° C. infirst-strand buffer comprising 50 mM Tris-HCl, pH 8.3; 75 mM KCl, 3 mMMgCl₂, 5 mM DTT, 1 mM dNTPs, and 20 U of RNase inhibitor, to which 25 Uof MULV reverse transcriptase was added. Viral RNAs were isolated fromconcentrated virion preparations derived from crHIV co-transfectedcultures. Cells and debris were removed by centrifugation at 2,000×g for15 min at 4° C. Virus was then concentrated by ultracentrifugation at30,000×g for 4 hr at 4° C. Viral RNAs were then isolated from the viralpellets using TriZol™ as previously described.

Viral RNAs were isolated from wild-type, HIV-transfected and crHIV-1.11co-transfected culture supernatants during the late stages of viralgrowth (i.e., wild-type HIV transfected cultures at day +17, crHIV-1.11co-transfected cultures at day +23). The virions in these culturescontained both wild-type HIV and crHIV genomic RNAs. Full-length,primer-extended cDNA was observed in both wild-type HIV transfected andcrHIV-1.11 co-transfected cultures. No smaller cDNAs, which would haveresulted from U5 RNA cleavage, were detected, despite extensiveprimer-extension analysis. Thus, the decrease in infectious wild-typeHIV titers is not due to intraviral cleavage of wild-type HIV RNAs, butto their numerical displacement by crHIV RNAs within progeny virions.

These results, thus, indicate that the method described herein can beemployed to displace wild-type genomes, such as HIV genomes and othergenomes, from progeny virions, using the conditionally replicatingvectors according to the invention.

Example 9

This example demonstrates that crHIV vectors can inhibit wild-type HIVreplication after challenge with plasmid or recombinant crHIV-1.111virus.

Jurkat cells were infected with stock HIV (clone pNL4-3) and were thenchallenged with either (1) plasmid DNA containing the crHIV-1.111construct or (2) recombinant crHIV-1.111 virus packaged in 293 cells,i.e., mutant crHIV-1.M (Nadlini et al., Science, 272, 263-267 (1996)).The cells were subjected to DLS lipid-mediated transfection (Thierry etal., PNAS, 92, 9742-9746 (1995)) or crHIV-mediated delivery. Viralreplication was measured by using the reverse transcriptase assay 12days after original infection with HIV. Wild-type positive controlcultures showed normal levels of wild-type HIV growth. When cellsinfected with wild-type HIV were challenged with mutant crHIV-1.M viaDLS-mediated transfection, wild-type HIV viral growth was unaffected. Incontrast, when cells infected with wild-type HIV were challenged withcrHIV-1.111, which encodes an anti-HIV ribozyme, via DLS-mediatedtransfection, wild-type HIV viral growth (i.e., replication) wassignificantly inhibited. Furthermore, when mutant crHIV-1.M waschallenged with wild-type HIV, wild-type HIV replication was unaffected.In contrast, when wild-type HIV was challenged with crHIV-1.111,wild-type HIV replication was significantly inhibited. The data showthat crHIV vectors can be used to inhibit significantly wild-type HIVreplication intracellularly.

Example 10

This example describes the use of conditionally replicating vectors inthe therapeutic treatment of cancer.

The conditionally replicating, cancer-treating, viral vector can beconstructed to be defective in its ability to replicate in normal cellsbecause it lacks a viral protein requisite for its replication. However,when this vector infects a cancerous cell, the unique properties of thecancerous cell provide a factor (e.g., preferably the same mutatedcellular protein that promotes the aberrant growth of cancerous cells)that facilitates the replication of the defective cancer-treatmentvector. Accordingly, this method differs from the method employed fortreatment of viral infection inasmuch as selective packaging of theviral vector does not occur, and instead, there is preferential lysis ofcancerous cells due to the packaging of progeny vector-derived virionsin the cell. However, the method is similar to the method employed forthe viral infections in that it can use a helper-virus expression vectorto selectively propagate the conditionally replicating vector incancerous cells. The vector and/or helper-virus expression vector can bemade to be responsive to tumor-specific factors, thereby facilitatingvector spread selectively in tumor cells.

Tumor-specific factors, which can be exploited in this method oftreatment, include, but are not limited to, those that act at the level:(1) of viral entry into cells (e.g., the presence of a tumor-specificreceptor that will allow a viral vector to selectively enter a cancerouscell, but not a normal cell); (2) of viral transcription (e.g., a mutantcancerous cell protein will allow a cancer-treatment vector totranscribe selectively its RNA in cancer cells, as opposed to normalcells; and (3) of viral maturation and release (e.g., mutant cancerouscellular proteins can allow the conditionally replicatingcancer-treatment vector to selectively mature, for instance, byassociation of the mutant cellular proteins with the viral proteins orgenome, and the resultant promotion of viral maturation and release).Accordingly, mutant proteins that exist in cancerous cells can interactwith viral proteins (or the genomic RNA or DNA) at many stages of theviral replication cycle. These interactions can be manipulated to createconditionally replicating cancer-treatment vectors, which are defectivein normal cells and can replicate in cancerous cells.

In particular, this method can be employed for the treatment of T-cellleukemia. T-cell leukemias are a severe form of cancer with a poorprognosis. Many of the leukemic T-cells are CD4⁺. Thus, an anti-T-cellleukemia-treating, conditionally replicating vector can be constructedusing wild-type HIV as the vector backbone. Inasmuch as HIV ostensiblyenters cells via the CD4 glycoprotein, this vector would act at thelevel of viral entry into cells.

The vector can be made into a cancer-treatment vector by introducingdeletions) into wild-type HIV, for example. The HIV genome can bemutated by producing it in its DNA form and conducting site-specificmutagenesis, as previously described. The method similarly can beemployed by complementing viral deficits with other tumor suppressormutations, or negative oncogenes, or by exploiting other tumor-specificfactors that interact with viral proteins. For example, the tat gene,which encodes a protein important for HIV replication, can be deleted.In the absence of Tat, HIV can no longer upregulate its expression,which is absolutely essential for HIV propagation. The Tat proteinfunctions by binding to the TAR RNA stem-loop structure, which isassociated with the HIV promoter, and is capable of upregulating HIVexpression by more than 100-fold. Thus, without Tat, the HIV-basedvector will not express HIV proteins, and will not propagate and killnormal (i.e., noncancerous) T-cells.

However, leukemic T-cells typically comprise a functionally alteredmolecule that is either mutated, overexpressed or silenced. This alteredstate of molecular function is not associated with normal cells. In itsnon-mutated state (but not its mutated state), this molecule functionsin the regulation of cell proliferation and/or apoptosis (programmedcell death). The changes associated with the mutated state can be usedto promote specifically the propagation of a conditionally replicatingviral vector. This could be done in the presence or absence of ahelper-virus expression vector. For example, the defect in Tat can becomplemented by a helper-expression vector that is driven of atumor-specific promoter, where the promoter is from a gene in leukemiccells that is overexpressed. Such a vector only can replicate inleukemic T-cells and not in normal cells. Viral expression andpropagation in leukemic T-cells would result in the lysis and death ofthe cells with nascent viral production. The vector could also carryadditional elements to promote cell killing (e.g., a sequence encoding atoxin, a cytokine or an antigen to promote immune targeting).

Other methods and strategies can similarly be employed in theconstruction of further conditionally replicating cancer-treatmentvectors.

Example 11

This example describes the development of second generation crHIVconstructs (cr2HIV), which have better propagation properties thancrHIV-1.111 vectors.

The second generation vectors enable increased production of crHIVparticles from crHIV-producing cells. The production of more crHIVparticles facilitates their spread and prevents wild-type HIV outgrowthin cultures. Lacking sequences encoding proteins that blocksuperinfection with wild-type HIV, the vectors contain all sequences ofthe native, wild-type HIV but do not encode the Tat gene. In place ofthe Tat gene is a triple anti-Tat ribozyme cassette ( SEQ ID NO:18!)made to the three different sites on the Tat gene. Also, the Tat splicesite was deleted so that the Tat ribozymes will selectively cleavegenomic wild-type HIV RNAs and not spliced wild-type HIV RNAs, whichcomplement for the defect in Tat and facilitate crHIV replication. Incontrast to the previous vectors, which do not encode proteins, otherthan, perhaps, a proteinaceous genetic antiviral agent, such as animmunogen, the second generation vectors encode, but only express, theseproteins in the presence of Tat. In a cell that contains both wild-typeHIV and crHIV genomes, crHIVs genomes will not only be selectivelypackaged, but many more virions will be produced than from crHIV-1.111cells, since the structural proteins are produced not only fromwild-type HIV, but from crHIV genomes as well. Accordingly, the vectoris conferred with a selective advantage for propagation, since it notonly is producing virions from wild-type HIV templates but also fromcrHIV templates.

The second generation vectors are also characterized by comprising orencoding ribozymes, the catalytic domains of which target regions otherthan those in the vector, itself. In contrast to crHIV-1.111, whichcomprises or encodes ribozymes targeted to the U5 region of the HIVleader sequence, which necessitated the incorporation of modified U5sequences into the leader of the crHIV vector, the ribozymes of thesecond generation vectors target regions that are not in the vector,itself, thereby eliminating the need to modify the sequence of thevector. This reduces the possibility that resistant HIVs could form byrecombination of wild-type HIV with modified crHIV U5 sequences. Thus,recombination of wild-type HIV with crHIV sequences would provide nobenefit to the wild-type HIV; incorporation of ribozyme sequences intowild-type HIV would only be detrimental to wild-type HIV.

The second generation vectors are further characterized by theincorporation of a number of different ribozymes, each of which istargeted to a different site, to reduce the possibility of wild-type HIVfrom forming ribozyme-resistant mutants.

In a further improvement of the vector system for the purposes of asafe, conditionally replicating vaccine, the "helper-vector" constructcan be further improved by adding genetic elements/factors thatspecifically facilitate crHIV replication and spread in a safe mannerOne embodiment is the introduction of ribozymes into the helper-vectorto prevent its genetic recombination with the vector to producewild-type virus. Thus, the above cr2HIV vector can be complemented witha Tat helper-expression vector to facilitate its spread. By insertinganti-HIV ribozymes into the helper-expression vector, the chance forrecombination is minimized because an encounter of the vector withhelper-vector RNA would result in their mutual scission and destruction.Therefore, the helper-expression vector can be modified in a number ofways to aid a particular prophylactic or therapeutic strategy.Accordingly, cr2HIV vectors have utility as vaccines against HIV sincethey (1) replicate and, thus, persistently stimulate the host's immuneresponse and (2) allow the host to recognize diverse epitopes, sincethey are derived from HIV and change antigenically.

All of the references cited herein, including patents, patentapplications, and publications, are hereby incorporated in theirentireties by reference.

While this invention has been described with an emphasis upon preferredembodiments, it will be apparent to those of ordinary skill in the artthat variations in the preferred embodiments can be prepared and usedand that the invention can be practiced otherwise than as specificallydescribed herein. The present invention is intended to include suchvariations and alternative practices. Accordingly, this inventionincludes all modifications encompassed within the spirit and scope ofthe invention as defined by the following claims.

    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 17    (2) INFORMATION FOR SEQ ID NO:1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 39 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    GTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATC39    (2) INFORMATION FOR SEQ ID NO:2:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 39 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    GTGTGCCCACCTGTTGTGTGACTCTGGCAGCTAGAGAAC39    (2) INFORMATION FOR SEQ ID NO:3:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 40 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (other nucleic acid)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    CACACAACACTGATGAGGCCGAAAGGCCGAAACGGGCACA40    (2) INFORMATION FOR SEQ ID NO:4:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 40 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (other nucleic acid)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    ATCTCTAGTCTGATGAGGCCGAAAGGCCGAAACCAGAGTC40    (2) INFORMATION FOR SEQ ID NO:5:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 39 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:    GTGTGCCCGCCTGTTGTGTGACTCTGGTAACTAGAGATC39    (2) INFORMATION FOR SEQ ID NO:6:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 39 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:    GTGTGCCCGTCTGTTGTGTGACTCTGGCAACTAGAGATC39    (2) INFORMATION FOR SEQ ID NO:7:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 27 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (other nucleic acid)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:    TGTGACGTCGACCACACAACACTGATG27    (2) INFORMATION FOR SEQ ID NO:8:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 33 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (other nucleic acid)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:    TGTGACGTCGACTCTAGATGTGCCCGTTTCGGC33    (2) INFORMATION FOR SEQ ID NO:9:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 31 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (other nucleic acid)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:    GGTTAAGCTTGAATTAGCCCTTCCAGTCCCC31    (2) INFORMATION FOR SEQ ID NO:10:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 31 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (other nucleic acid)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:    GGTTGGATCCGGGTGGCAAGTGGTCAAAAAG31    (2) INFORMATION FOR SEQ ID NO:11:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 38 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (other nucleic acid)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:    CGGATCCACGCGTGTCGACGAGCTCCCATGGTGATCAG38    (2) INFORMATION FOR SEQ ID NO:12:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 31 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (other nucleic acid)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:    GGTTAAGCTTGTCGCCGCCCCTCGCCTCTTG31    (2) INFORMATION FOR SEQ ID NO:13:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 112 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (other nucleic acid)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:    AAGCTTGCCTTGAGTGCTCAAAGTAGTGTGTGCCCACCTGTTGTGTGACTCTGGCAGCTA60    GAGATCCCACAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTGGCGCC112    (2) INFORMATION FOR SEQ ID NO:14:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 39 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:    GTGTGCCCNNCTGTTGTGTGACTCTGGNANCTAGAGANC39    (2) INFORMATION FOR SEQ ID NO:15:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 39 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:    GTGTGCCCATCTGTTGTGTGACTCTGGTAACTAGAGATC39    (2) INFORMATION FOR SEQ ID NO:16:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 39 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:    GTGTGCCCGTCTGTTGTGTGACTCTGGTAGCTAGAGATC39    (2) INFORMATION FOR SEQ ID NO:17:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 152 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (other)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:    GATCGAATTCCTGCTATGTTCTGATGAGTCCGAAAGGACGAAACACCCATTTCCCGGGTT60    TAGGATCCTGATGAGCGGAAAGCCGCGAAACTGGCTCCGGCCGTTTTAGGCTCTGATGAG120    CTGGAAACAGCGAAACTTCCTGGTCGACGATC152    __________________________________________________________________________

What is claimed is:
 1. A method of making a product humaninmunodeficiency viral vector, which method comprises:(a) obtaining astarting vector, which is derived from a wild-type strain of humanimmunodeficiency virus and which replicates only in a host cell that ispermissive for replication of said starting vector; and (b)incorporating into the starting vector of (a) a nucleic acid sequence,which comprises or encodes, in which case it also expresses, a geneticantiviral agent, and thereby, or in addition thereto, modifying thestarting vector of (a) to obtain a product vector, which is resistant tosaid genetic antiviral agent, wherein said product vector is selectivelypackaged into progeny virions over (i) a wild-type strain of humanimmunodeficiency virus, which is sensitive to said genetic antiviralagent, or (ii) a helper virus or a helper vector for replication of ahuman immunodeficiency viral vector, which is sensitive to said geneticantiviral agent, when (i) said wild-type strain of humanimmunodeficiency virus or (ii) said helper virus or said helper vectorfor replication of a human immunodeficiency viral vector is present insaid host cell.
 2. The method of claim 1, wherein said genetic antiviralagent is an antisense molecule.
 3. The method of claim 1, wherein saidgenetic antiviral agent is a ribozyme.
 4. The method of claim 3, whereinstep (b) comprises:(i) incorporating into the starting vector of (a),wherein said vector is DNA, a nucleotide sequence selected from thegroup consisting of SEQ ID NO:3 and SEQ ID NO:4; and (ii) modifying thestarting vector of (a) to comprise a nucleotide sequence of SEQ IDNO:14, in which at least one N is mutated.
 5. The method of claim 4,wherein step (b) comprises;(i) incorporating into the starting vector of(a) SEQ ID NO:3; and (ii) modifying the starting vector of (a) tocomprise a nucleotide sequence selected from the group consisting of SEQID NOS:2, 5 and
 15. 6. The method of claim 4, wherein step (b)comprises:(i) incorporating into the starting vector of (a) SEQ ID NO:4;and (ii) modifying the starting vector of (a) to comprises a nucleotidesequence selected from the group consisting of SEQ ID NOS:2, 6 and 16.7. The method of claim 3, wherein step (b) comprises:(i) incorporatinginto the starting vector of (a), wherein said vector is RNA, anucleotide sequence encoded by a nucleotide sequence selected from thegroup consisting of SEQ ID NO:3 and SEQ ID NO:4; and (ii) modifying thestarting vector of (a) to comprise a nucleotide sequence encoded by anucleotide sequence of SEQ ID NO:14, in which at least one N is mutated.8. The method of claim 7, wherein step (b) comprises:(i) incorporatinginto the starting vector of (a) the nucleotide sequence encoded by SEQID NO:3; and (ii) modifying the starting vector of (a) to comprise anucleotide sequence encoded by a nucleotide sequence selected from thegroup consisting of SEQ ID NOS:2, 5 and
 15. 9. The method of claim 7,wherein step (b) comprises:(i) incorporating into the vector of (a) thenucleotide sequence encoded by SEQ ID NO:4;and (ii) modifying thestarting vector of (a) to comprise a nucleotide sequence encoded by anucleotide sequence selected from the group consisting of SEQ ID NOS:2,6 and
 16. 10. The method of claim 3, wherein said ribozyme is a ribozymecassette comprising one, two or multiple ribozymes.
 11. The method ofclaim 10, wherein said ribozyme cassette comprises multiple ribozymes.12. The method of claim 11, wherein said ribozyme cassette comprisesthree ribozymes.
 13. A conditionally replicating human immunodeficiencyviral vector, wherein said vector is selected from the group consistingof crHIV-1.1, crHIV-1.11, crHIV- 1.12, and crHIV-1.111.
 14. A method ofmodifying a vector, which method comprises:(a) obtaining a humaninmunodeficiency viral vector, wherein said vector is DNA; and (b)introducing into the vector of (a) a nucleotide sequence selected fromthe group consisting of SEQ ID NOS:2, 5, 6, 15 and
 16. 15. An isolatedand purified RNA molecule comprising a nucleotide sequence encoded by anucleotide sequence selected from the group consisting of SEQ ID NOS:2,5, 6, 15 and 16.